xCELLigence Cardio Symposium 2014

Cardiotoxicity QT Prolongation and Arrhythmia

Prediction Using iCell Cardiomyocytes Past

Present and Future

Blake Anson PhD

Oct 18 2014

Outline

Proarrhythmia Testing

- moving from single channel to holistic

assessments

Cardiotoxicity Testing

- iCell Cardiomyocytes and xCelligence

RTCA

Drug Discovery and Population

- Disease modeling and incorporating

populations

Key manufacturing components

3

In-vitro detection of proarrhythmia

The road to in-vitro

proarrhythmia testinghellip

hellip started in a fly

4

Drug ndashinduced Electrophysiological Aberrations

not a new phenomenon

Quinidine Syncope and Delayed

Repolarization SyndromesReynolds E and Vander Ark C M

Modern Concepts of Cardiovascular Disease 45117-

122 1976

Davies et al BMJ 1989298

Wyosowski and Bacsanyi NEJM 1995335

Astemizole-induced Arrhythmmia

From Vorperian et al JACC 199615

hellipbut took on a new meaning when

caused by non-cardiac compounds

5

Fruit flies provided insight to arrhythmia

Leg shaking

EAG mutant

(ether-a-go-go)

Wild

type

EAG Gene Ether-sedated Drosophila (Fruit Flies)

B Ganetzky

6

hERG is a member of the EAG

superfamily of K+ channels

Library Screen(s)

Hippocampal mRNA

Hit

Analysis

Heterologous

Expression

hERG ndash human ether a

go-go related gene

The hERG gene is linked

to Long QT Syndrome

EAG ndashether a go-go

gene sequence

The hERG gene

encdes lKr

7

DIA

Arrhythmogenic drugs block hERG channels and

prolong the cardiac AP

Zhou and January 1997

8

hERG and In-vitro Safety Assessments

Highly sensitive with

questionable specificity

9

Comprehensive in-vitro Proarrhythmia Testing

The Future

Comprehensive in-vitro Proarrhythmia

Assessment (CiPA)1 Assess effects on multiple individual ion

channels

2 Model effects (if any) on the ventricular

action potential and proarrhythmia

3 Verify conclusions with cardiomyocyte

recordings

httpwwwilsiextraorghesisciencecardiaccipa

Sager et al American Heart Journal (2014)

10

Pro-arrhythmia Testing and Beyond

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

Outline

Proarrhythmia Testing

- moving from single channel to holistic

assessments

Cardiotoxicity Testing

- iCell Cardiomyocytes and xCelligence

RTCA

Drug Discovery and Population

- Disease modeling and incorporating

populations

Key manufacturing components

3

In-vitro detection of proarrhythmia

The road to in-vitro

proarrhythmia testinghellip

hellip started in a fly

4

Drug ndashinduced Electrophysiological Aberrations

not a new phenomenon

Quinidine Syncope and Delayed

Repolarization SyndromesReynolds E and Vander Ark C M

Modern Concepts of Cardiovascular Disease 45117-

122 1976

Davies et al BMJ 1989298

Wyosowski and Bacsanyi NEJM 1995335

Astemizole-induced Arrhythmmia

From Vorperian et al JACC 199615

hellipbut took on a new meaning when

caused by non-cardiac compounds

5

Fruit flies provided insight to arrhythmia

Leg shaking

EAG mutant

(ether-a-go-go)

Wild

type

EAG Gene Ether-sedated Drosophila (Fruit Flies)

B Ganetzky

6

hERG is a member of the EAG

superfamily of K+ channels

Library Screen(s)

Hippocampal mRNA

Hit

Analysis

Heterologous

Expression

hERG ndash human ether a

go-go related gene

The hERG gene is linked

to Long QT Syndrome

EAG ndashether a go-go

gene sequence

The hERG gene

encdes lKr

7

DIA

Arrhythmogenic drugs block hERG channels and

prolong the cardiac AP

Zhou and January 1997

8

hERG and In-vitro Safety Assessments

Highly sensitive with

questionable specificity

9

Comprehensive in-vitro Proarrhythmia Testing

The Future

Comprehensive in-vitro Proarrhythmia

Assessment (CiPA)1 Assess effects on multiple individual ion

channels

2 Model effects (if any) on the ventricular

action potential and proarrhythmia

3 Verify conclusions with cardiomyocyte

recordings

httpwwwilsiextraorghesisciencecardiaccipa

Sager et al American Heart Journal (2014)

10

Pro-arrhythmia Testing and Beyond

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

3

In-vitro detection of proarrhythmia

The road to in-vitro

proarrhythmia testinghellip

hellip started in a fly

4

Drug ndashinduced Electrophysiological Aberrations

not a new phenomenon

Quinidine Syncope and Delayed

Repolarization SyndromesReynolds E and Vander Ark C M

Modern Concepts of Cardiovascular Disease 45117-

122 1976

Davies et al BMJ 1989298

Wyosowski and Bacsanyi NEJM 1995335

Astemizole-induced Arrhythmmia

From Vorperian et al JACC 199615

hellipbut took on a new meaning when

caused by non-cardiac compounds

5

Fruit flies provided insight to arrhythmia

Leg shaking

EAG mutant

(ether-a-go-go)

Wild

type

EAG Gene Ether-sedated Drosophila (Fruit Flies)

B Ganetzky

6

hERG is a member of the EAG

superfamily of K+ channels

Library Screen(s)

Hippocampal mRNA

Hit

Analysis

Heterologous

Expression

hERG ndash human ether a

go-go related gene

The hERG gene is linked

to Long QT Syndrome

EAG ndashether a go-go

gene sequence

The hERG gene

encdes lKr

7

DIA

Arrhythmogenic drugs block hERG channels and

prolong the cardiac AP

Zhou and January 1997

8

hERG and In-vitro Safety Assessments

Highly sensitive with

questionable specificity

9

Comprehensive in-vitro Proarrhythmia Testing

The Future

Comprehensive in-vitro Proarrhythmia

Assessment (CiPA)1 Assess effects on multiple individual ion

channels

2 Model effects (if any) on the ventricular

action potential and proarrhythmia

3 Verify conclusions with cardiomyocyte

recordings

httpwwwilsiextraorghesisciencecardiaccipa

Sager et al American Heart Journal (2014)

10

Pro-arrhythmia Testing and Beyond

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

4

Drug ndashinduced Electrophysiological Aberrations

not a new phenomenon

Quinidine Syncope and Delayed

Repolarization SyndromesReynolds E and Vander Ark C M

Modern Concepts of Cardiovascular Disease 45117-

122 1976

Davies et al BMJ 1989298

Wyosowski and Bacsanyi NEJM 1995335

Astemizole-induced Arrhythmmia

From Vorperian et al JACC 199615

hellipbut took on a new meaning when

caused by non-cardiac compounds

5

Fruit flies provided insight to arrhythmia

Leg shaking

EAG mutant

(ether-a-go-go)

Wild

type

EAG Gene Ether-sedated Drosophila (Fruit Flies)

B Ganetzky

6

hERG is a member of the EAG

superfamily of K+ channels

Library Screen(s)

Hippocampal mRNA

Hit

Analysis

Heterologous

Expression

hERG ndash human ether a

go-go related gene

The hERG gene is linked

to Long QT Syndrome

EAG ndashether a go-go

gene sequence

The hERG gene

encdes lKr

7

DIA

Arrhythmogenic drugs block hERG channels and

prolong the cardiac AP

Zhou and January 1997

8

hERG and In-vitro Safety Assessments

Highly sensitive with

questionable specificity

9

Comprehensive in-vitro Proarrhythmia Testing

The Future

Comprehensive in-vitro Proarrhythmia

Assessment (CiPA)1 Assess effects on multiple individual ion

channels

2 Model effects (if any) on the ventricular

action potential and proarrhythmia

3 Verify conclusions with cardiomyocyte

recordings

httpwwwilsiextraorghesisciencecardiaccipa

Sager et al American Heart Journal (2014)

10

Pro-arrhythmia Testing and Beyond

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

5

Fruit flies provided insight to arrhythmia

Leg shaking

EAG mutant

(ether-a-go-go)

Wild

type

EAG Gene Ether-sedated Drosophila (Fruit Flies)

B Ganetzky

6

hERG is a member of the EAG

superfamily of K+ channels

Library Screen(s)

Hippocampal mRNA

Hit

Analysis

Heterologous

Expression

hERG ndash human ether a

go-go related gene

The hERG gene is linked

to Long QT Syndrome

EAG ndashether a go-go

gene sequence

The hERG gene

encdes lKr

7

DIA

Arrhythmogenic drugs block hERG channels and

prolong the cardiac AP

Zhou and January 1997

8

hERG and In-vitro Safety Assessments

Highly sensitive with

questionable specificity

9

Comprehensive in-vitro Proarrhythmia Testing

The Future

Comprehensive in-vitro Proarrhythmia

Assessment (CiPA)1 Assess effects on multiple individual ion

channels

2 Model effects (if any) on the ventricular

action potential and proarrhythmia

3 Verify conclusions with cardiomyocyte

recordings

httpwwwilsiextraorghesisciencecardiaccipa

Sager et al American Heart Journal (2014)

10

Pro-arrhythmia Testing and Beyond

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

6

hERG is a member of the EAG

superfamily of K+ channels

Library Screen(s)

Hippocampal mRNA

Hit

Analysis

Heterologous

Expression

hERG ndash human ether a

go-go related gene

The hERG gene is linked

to Long QT Syndrome

EAG ndashether a go-go

gene sequence

The hERG gene

encdes lKr

7

DIA

Arrhythmogenic drugs block hERG channels and

prolong the cardiac AP

Zhou and January 1997

8

hERG and In-vitro Safety Assessments

Highly sensitive with

questionable specificity

9

Comprehensive in-vitro Proarrhythmia Testing

The Future

Comprehensive in-vitro Proarrhythmia

Assessment (CiPA)1 Assess effects on multiple individual ion

channels

2 Model effects (if any) on the ventricular

action potential and proarrhythmia

3 Verify conclusions with cardiomyocyte

recordings

httpwwwilsiextraorghesisciencecardiaccipa

Sager et al American Heart Journal (2014)

10

Pro-arrhythmia Testing and Beyond

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

7

DIA

Arrhythmogenic drugs block hERG channels and

prolong the cardiac AP

Zhou and January 1997

8

hERG and In-vitro Safety Assessments

Highly sensitive with

questionable specificity

9

Comprehensive in-vitro Proarrhythmia Testing

The Future

Comprehensive in-vitro Proarrhythmia

Assessment (CiPA)1 Assess effects on multiple individual ion

channels

2 Model effects (if any) on the ventricular

action potential and proarrhythmia

3 Verify conclusions with cardiomyocyte

recordings

httpwwwilsiextraorghesisciencecardiaccipa

Sager et al American Heart Journal (2014)

10

Pro-arrhythmia Testing and Beyond

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

8

hERG and In-vitro Safety Assessments

Highly sensitive with

questionable specificity

9

Comprehensive in-vitro Proarrhythmia Testing

The Future

Comprehensive in-vitro Proarrhythmia

Assessment (CiPA)1 Assess effects on multiple individual ion

channels

2 Model effects (if any) on the ventricular

action potential and proarrhythmia

3 Verify conclusions with cardiomyocyte

recordings

httpwwwilsiextraorghesisciencecardiaccipa

Sager et al American Heart Journal (2014)

10

Pro-arrhythmia Testing and Beyond

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

9

Comprehensive in-vitro Proarrhythmia Testing

The Future

Comprehensive in-vitro Proarrhythmia

Assessment (CiPA)1 Assess effects on multiple individual ion

channels

2 Model effects (if any) on the ventricular

action potential and proarrhythmia

3 Verify conclusions with cardiomyocyte

recordings

httpwwwilsiextraorghesisciencecardiaccipa

Sager et al American Heart Journal (2014)

10

Pro-arrhythmia Testing and Beyond

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

10

Pro-arrhythmia Testing and Beyond

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

11

Interrogating BiologyElectrical and Mechanical Activity

Cardiomyocyte Activity

Electrical biochemical and mechanical

Electrical

Biochemical

Mechanical

Three main areas need to be

considered for cardiotoxicity

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

12

Predicting ProarrhythmiaLabel Free Impedance Measurements

iCell Cardiomyocytes provide a more predictive tool for detecting proarrhythmia

Greater Predictivity

~120 Compounds

gt90 -- QT prediction

gt82 -- arrhy prediction

Qualitative Assessment

Guo et al 2011

Guo et al 2013

Relevant biology and metrics leads to

greater predictivity

Expanded dataset

o ~120 compounds

Fine tune metrics

o Include beat rate

atypical beats

onset of IB20

o Use concentration

thresholds or IB20

rank ordering

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

13

KI-induced Cardiotoxicity

Deconvoluting the problem

S Lamore AstraZeneca

iCell Cardiomyocytes provide a predictive tool for detecting KI toxicity

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

15

Parameter IonOptix

sensitivity 83

specificity 84

accuracy 82

pos predict 90

neg predict 76

Parameter Impedance3

sensitivity 90

specificity 74

accuracy 84

pos predict 85

neg predict 82

IonOptix

Good to excellent validation parameters

Primary culture from dog heart

Low throughput

Conventional Interrogation

Screening with iCell Cardiomyocytes

1 AR Harmer Tox App Pharm 2012

iCell Cardiomyocytes provide a predictive

model for detecting contractility

2 C Scott (Tox Sci 2014 )

49 compound validation set with actives and inactives

xCelligence RTCA

Good to excellent assay parameters2

Human cardiomyocytes

Medium to high throughput

Detecting Effects on ContractilityMoving to higher throughput predictive detection

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

16

iCell Cardiomyocytes and xCelligence RTCA Predictive solutions for multi-modal cardiotoxicity

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

17

Disease ModelingCase 1 Cardiac Hypertrophy

Cellular and Molecular Markers

bull Increased cell size

bull Enhanced protein synthesis sarcomeric organization

bull Re-activation of the fetal gene program (BNP ANP etc)

Normal

Diseased

Lister K et al Cardiovasc Res 200670555-565

Ce

ll S

ize

Ac

tin

Re

org

Untreated + PE

Glenn D et al Hypertension 200953549-555

Kuwahara K et al J Pharmacol Sci 2012119198-203

Protein Synthesis BNP Expression

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

18

In-Vitro Recapitulation of HypertrophyiCellreg Cardiomyocytes

Control

ET-1 (10 nM)

-14 -13 -12 -11 -10 -9 -81000

1100

1200

1300

1400

1500

Log [ET-1] (M)

To

tal A

rea (

m2)

Control

+ET-1 (10 nM)

Control

+ET-1 (10 nM)

Cell SizeCytoskeletal

Rearrangements

Fetal Gene

Expression

iCell Cardiomyocytes exhibit classic hallmarks of cardiac hypertrophy

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

19

iCell Cardiomyocyte HypertrophyRelevance

Aggarwal et al Plos One 2014

Hypertrophic iCell Cardiomyocytes share similarities with cardiac

samples from LVH patients

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

20

Case 2 Diabetic Cardiac MyopathyEnvironmental Induction

Application of a diabetic medium (ET-1 cortisol glucose) to iCell CMs induces a hypertrophic phenotype

Increases in

bull Cell and nuclear size

bull Glycolysis

bull Cytoskeletal disorganization

bull Lipid accumulation

bull ROS Accumulation

Drawnel 2014 in press

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

21

RO

S P

rod

ucti

on

iPSC-CMs from diabetic patients exhibit markers of hypertrophy under basal conditions

Cytoskeletal

disorganization

Lipid

Accumulation

Oxidative

StressCompounds have been identified that revert the

diabetic phenotype present in the iPSC-CMs

S iCell CMs

SP MyCells Di-CM appearance gt 15 yrs post

diabetes onset

FP MyCells Di-CM appearance lt 5 yrs post

diabetes onset

Case 3 Diabetic Cardiac MyopathyEnvironmental Induction

Drawnel 2014 in press

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

22

MyCell MYH7

R403Q CMiCell CM

98 96

cT

nT

NPPB 5

ACTA1 4

DUSP4 3

ACTC1 2

ACTN1 1

CREB5 0

MYH7 -1

NPPA -2

MYH6 -3

TRIM63 -4

ADM -5

FBXO32

PDCD4

Relative Expression

ET-1

induced

iCell CM

MyCell

MYH7

R403Q CM

Cell Type ViabilityPlating

Efficiency

iCell Cardiomyocytes (CM) 79 56

MyCell MYH7 R403Q CM 76 45

MYH7 R403Q hypertrophic cardiomyopathy

(A) ndash (C) Manufacture of custom lines is similar to

catalog products

(D) MYH7 R403Q show signs of cardiac hypertrophy

under basal conditions

A

B

C

D

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

hIPSC-Cardiomyocytes mimic induced and innate disease models

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

23

iCell CM MYH7 R403Q CM

iCell and MYH7 R403QCMs differ in

basal BNP expression but respond

similarly to ET-1 induction

BNP DAPI 10X image in 384-well plate

iCell and MYH7 R403QCMs both

respond to pathway inhibition

Case study 4 Induced disease modelsMYH7-R403Q linked hypertrophic cardiomyopathy

MYH7 R403Q CMsbull Carry a hypertrophic phenotype

bull Are inducible via ET1

bull React to phenotypic reversion screens

bull Represent a disease-based screening model

hIPSC-Cardiomyocytes mimic induced and innate disease models

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

24

The Power of IPSC Technology

hellippopulations

What abouthellip

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

25

StandardizationManufacturing Benchmarks

NHLBI Next Generation Genetic

Association Studies(RFA-HL-11-066)

250 patient samples - HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes from

all 250 individuals

Induce hypertrophy phenotype perform

molecular analyses

Correlate GWAS findings with in vitro

phenotype

Scale-Out

Manufacturing

bull 1000rsquos of individuals

bull Billions of cells

Scale-Up

Manufacturing

bull Quality

bull Quantity

bull Purity

CDI Manufacturing Benchmarks (cells per day gt95 purity)

2 billion iPS cells

1 billion cardiomyocytes

1 billion neurons

05 billion endothelial cells

04 billion hepatocytes

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

26

NHLBI Next Generation Genetic

Association Studies (RFA-HL-11-066)

250 patient samples ndash HyperGEN cohort

GWAS ndash Left Ventricular Hypertrophy (LVH)

Derive iPS cells and cardiomyocytes

Induce hypertrophy perform molecular analyses

Correlate GWAS findings with in vitro phenotype

Progress as of July 2014

250 donors reprogrammed

Differentiation protocol optimized to work robustly across all lines

128 iPS cell lines (1 per donor) are differentiated or in progress

Cardiomyocytes from 89 donors cryopreserved amp all pass QC

20 batches of cardiomyocytes are in currently being tested in

hypertrophy assays

Initial data show Et-1 EC50 correlation with progression of disease (Uli Broeckel MCOW)

Progress ReportPopulation genomics and left ventricular hypertrophy

CDIrsquos iPSC technology is enabling population studies

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

27

CIRM AwardiPS Cell Manufacture amp Banking

California Institute for Regenerative Medicine (CIRM)

Human iPS Cell Initiative ndash 3 Awards

Sample Collection (7 awardees)

iPS Cell Derivation (CDI)

iPS Cell Banking (Coriell CDI primary subcontractor)

iPS Cell Derivation

3000 donors (healthy amp disease phenotypes)

3 iPS cell clones per donor

Disease categories epilepsy autism cerebral palsy cardiomyopathy Alzheimerrsquos

disease eye diseases hepatitis (HCV) non-alcoholic steatohepatitis (NASH)

pulmonary fibrosis

Derived from peripheral blood (preferred) or skin fibroblasts

Episomal ldquofootprint-freerdquo method

CDI ndash Coriell Partnership

Extensive collaboration to bring together expertise in electronic record-keeping

sample tracking iPS cell derivation amp characterization cell banking amp distribution

Joint facility located within the Buck Institute Novato CA

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

28

Will this potential be

realized

Yes

But it is not easy

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

29

CDI CommitmentQuality Quantity Purity

Quality

Quantity Purity

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Sufficient to support HTP drug screening

and safety testing

Currently 1Bn iCell Cardiomyocytesday

Ce

ll P

uri

ty

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

QMS Framework Overview

Key Systems Objectives

QAQC Compliance and product consistency

Standard Operating Procedures Consistent procedures

CalibrationQualVal Equipmentfacilitiesprocesses fit for intended use

Change Management Changes are documented assessed for risk and tested

CAPA Report correct and prevent product quality issues

Supplier Qual amp Mgmt Quality and reliability of raw materials

Materials Management Control trace and monitor stock inventory

Training Education and proficiency

Complaint Handling Customer satisfaction and continuous improvement

New Product Introduction Improve likelihood that product meets market need

An ISO GMP hybrid QMS system ensures customer safety and satisfaction

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

31

1 Nakamura Y1 Matsuo J (2014) Assessment of testing methods for

drug-induced repolarization delay and arrhythmias in an iPS cell-

derived cardiomyocyte sheet multi-site validation study J

Pharmacol Sci 124(4)494-501

2 Eldridge S Guo L et al (2014) Examining the Protective Role of

ErbB2 Modulation in Human Induced Pluripotent Stem Cell-

Derived Cardiomyocytes Toxicol Sci 2014 Jul 23 pii kfu150

[Epub ahead of print]

3 Kolaja K (2014) Stem cells and stem cell-derived tissues and their

use in safety assessment J Biol Chem 2014 Feb 21289(8)4555-

61

4 Uesugi M Ojima A et al (2014) Low-density plating is sufficient to

induce cardiac hypertrophy and electrical remodeling in highly

purified human iPS cell-derived cardiomyocytes J Pharmacol

Toxicol Methods 69(2)177-88

5 Cameron BJ Gerry AB et al (2013) Identification of a Titin-

derived HLA-A1-presented peptide as a cross-reactive target for

engineered MAGE A3-directed T cells Sci Transl Med

5(197)197ra103

6 Carlson C Koonce C et al (2013) Phenotypic screening with

human iPS cell-derived cardiomyocytes HTS-compatible assays

for interrogating cardiac hypertrophy J Biomol Screen

18(10)1203-11

7 Doherty K Wappel R et al (2013) Multiparameter in vitro toxicity

testing of crizotinib sunitinib erlotinib and nilotinib in human

cardiomyocytes Toxicol Appl Pharmacol 272(1)245-55

8 Fine M Lu F et al (2013) Human Induced Pluripotent Stem Cell-

derived Cardiomyocytes for Studies of Cardiac Ion Transporters

Am J Physiol Cell Physiol 305(5)C481-91

9 Guo L Coyle l et al (2013) Refining the Human iPSC-

Cardiomyocyte Arrhythmic Risk Assessment Model Toxicol Sci

136(2)581-94

10 Harris K Aylott M et al (2013) Comparison of

Electrophysiological Data from Human Induced Pluripotent Stem

Cell Derived Cardiomyoyctes (hiPSC-CMs) to Functional Pre-

clinical Safety Assays Toxicol Sci 134(2)412-26

11 Ivashchenko CY1 Pipes GC et al (2013) Human-induced

pluripotent stem cell-derived cardiomyocytes exhibit temporal

changes in phenotype Am J Physiol Heart Circ Physiol

305(6)H913-22

12 Jehle J Ficker E et al (2013) Mechanisms of Zolpidem-induced

Long QT Ayndrome Acute Inhibition of Recombinant hERG K+

Channels and Action Potential Prolongation in Human

Cardiomyocytes Derived from Induced Pluripotent Stem Cells

British J Pharm 1681215-29

13 Puppala D Collis LP et al (2013) Comparative Gene Expression

Profiling in Human Induced Pluripotent Stem Cell Derived

Cardiocytes and Human and Cynomolgus Heart Tissue Toxicol

Sci 131292-301

14 Rao C Prodromakis T et al (2013) The effect of microgrooved

culture substrates on calcium cycling of cardiac myocytes

derived from human induced pluripotent stem cells Biomaterials

34(10)2399-411

15 Schweikart K Guo L et al (2013) The Effects of Jaspamide on

Human Cardiomyocyte Function and Cardiac Ion Channel Activity

Toxicol in Vitro 27745-51

16 Sirenko O Crittenden C et al (2013) Multiparameter In Vitro

Assessment of Compound Effects on Cardiomyocyte Physiology

Using iPS Cells J Biomol Screening 1839-53

17 Sirenko O Cromwell EF et al (2013) Assessment of beating

parameters in human induced pluripotent stem cells enables

quantitative in vitro screening for cardiotoxicity Toxicol Appl

Pharmacol 273(3)500-07

18 Babiarz JE Ravon M et al (2012) Determination of the Human

Cardiomyocyte mRNA and miRNA Differentiation Network by

Fine-scale Profiling Stem Cells Dev 211956-65

19 Cerignoli R Charlot D et al (2012) High Throughput Measurement

of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-

derived Cardiomyocytes by Kinetic Image Cytometry

J Pharmacol Toxicol Methods 66246-256

20 Lee P Kloss M et al (2012) Simultaneous Voltage and Calcium

Mapping of Genetically Purified Human Induced Pluripotent Stem

Cell-derived Cardiac Myocyte Monolayers Circ Res 1101556-63

21 Mioulane M Foldes G et al (2012) Development of High Content

Imaging Methods for Cell Death Detection in Human Pluripotent

Stem Cell-derived Cardiomyocytes J of Cardiovasc Trans Res

5593-604

22 Rana P Anson BD et al (2012) Characterization of Human-

induced Pluripotent Stem Cell-derived Cardiomyocytes

Bioenergetics and Utilization in Safety Screening Toxicol Sci

130117-31

23 Reynolds JG Geretti E et al (2012) HER2-targeted Liposomal

Doxorubicin Displays Enhanced Anti-tumorigenic Effects without

Associated Cardiotoxicity Toxicol Appl Pharmacol 2621-10

24 Wei H Zhang G et al (2012) Hydrogen Sulfide Suppresses

Outward Rectifier Potassium Currents in Human Pluripotent Stem

Cell-Derived Cardiomyocytes Plos One 7(11)e50641

25 Zhi D Irvin MR et al (2012) Whole-exome Sequencing and an

iPSC-derived Cardiomyocyte Model Provides a Powerful Platform

for Gene Discovery in Left Ventricular Hypertrophy Frontiers in

Genetics 392

26 Cohen JD Babiarz JE et al (2011) Use of Human Stem Cell-

derived Cardiomyocytes to Examine Sunitinib Mediated

Cardiotoxicity and Electrophysiological Alterations Toxicol Appl

Pharmacol 25774-83

27 Guo L Qian JY et al (2011) The Electrophysiological Effects of

Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and

in Guinea Pig Isolated Hearts Cell Physiol Biochem 27453-462

28 Guo L Abrams RM et al (2011) Estimating the Risk of Drug-

induced Proarrhythmia Using Human Induced Pluripotent Stem

Cell-derived Cardiomyocytes Toxicol Sci 123281-289

29 Jonsson MKB Wang QD et al (2011) Impedance-based Detection

of Beating Rhythm and Proarrhythmic Effects of Compounds on

Stem Cell-derived Cardiomyocytes Assay and Drug Dev Tech 91-

11

30 Ma J Guo L et al (2011) High Purity Human-induced Pluripotent

Stem Cell-derived Cardiomyocytes Electrophysiological

Properties of Action Potentials and Ionic Currents Am J Physiol

Heart Circ Physiol 301H2006-H2017

iCell CardiomyocytesMarket Validation (82014)

~40 Peer-reviewed

Publications (102014)

bull Characterization

bull Toxicity testing

bull Disease modeling

32

Summary

Proarrhythmia Testing

- moving toward a cellular mechanistic approach that may take advantage of stem cell cardiomyocytes

Cardiotoxicity Testing

- iCell Cardiomyocytes and xCelligence RTCA provide predictive solutions

Drug Discovery and Population

- CDI products exhibit induced and innate disease phenotypes for drug discovery

Key manufacturing components

- Quality is king

33

Product Portfolio

2009 2010 2011 2012 2013

Essential 8 Medium

Episomal Reprogramming Kit

Vitronectin

iCell Products

iCell Cardiomyocytes

iCell Cardiac Progenitor Cells (New)

iCell Hematopoietic Progenitor Cells

iCell Endothelial Cells

iCell Hepatocytes

iCell Neurons

iCell Astrocytes

iCell DopaNeurons (New)

iCell Skeletal Myoblasts

MyCell Products

iPS Cell Reprogramming

iPS Cell Genetic Engineering

iPS Cell Differentiation

MyCell Disease and Diversity Panel (New)

2014

iCell Cardiomyocytes iCell

Endothelial Cells

MyCell Products

iCell

Hepatocytes

iCell Astrocytes

iCell

Hematopoietic

Progenitor

Cells

iCell Skeletal

Myoblasts

iCell Cardiac

Progenitor CellsiCell

DopaNeurons

iCell Neurons

33

Product Portfolio

2009 2010 2011 2012 2013

Essential 8 Medium

Episomal Reprogramming Kit

Vitronectin

iCell Products

iCell Cardiomyocytes

iCell Cardiac Progenitor Cells (New)

iCell Hematopoietic Progenitor Cells

iCell Endothelial Cells

iCell Hepatocytes

iCell Neurons

iCell Astrocytes

iCell DopaNeurons (New)

iCell Skeletal Myoblasts

MyCell Products

iPS Cell Reprogramming

iPS Cell Genetic Engineering

iPS Cell Differentiation

MyCell Disease and Diversity Panel (New)

2014

iCell Cardiomyocytes iCell

Endothelial Cells

MyCell Products

iCell

Hepatocytes

iCell Astrocytes

iCell

Hematopoietic

Progenitor

Cells

iCell Skeletal

Myoblasts

iCell Cardiac

Progenitor CellsiCell

DopaNeurons

iCell Neurons