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Human iPSC-derived cardiomyocytes, neurons, and hepatocytes;
2017_05_18
Blake Anson, PhD
Cells and solutions to improve toxicity testing relevance and predictivity.
CONFIDENTIAL 2May 18, 2017
Agenda
• Company Overview
• iCell Hepatocytes
• Extended culture time, 3D spheroids, increased
functionality
• iCell GlutaNeurons / iCell Neurons
• Seizurogenic assays
• Structural toxicity and Chemotherapy-induced
Neuropathy
• iCell Cardiomyocytes
• Ephys and oncology-related case studies
May 18, 2017 3
Company Overview
• Cellular Dynamics International (CDI) is a leader in production and application of human iPS cells and iPS cell-derived cell types
• Acquired by FUJIFILM (4/2015); International presence
Headquartered in Madison, WI (additional site in Novato, CA)
Application / Distribution sites in Japan, South Korea, and Tilburg
Local Sales and FAS support
• Currently employs ~175 total staff w/ >900yrs cumulative stem cell and differentiation experience
• >900 patents (owned or licensed)
• Portfolio includes off the shelf products, as well as, custom cell production and assay services.
May 18, 2017 4
FCDI Driving Expertise and Technology
Bringing relevant human biology and diversity to drug discovery
May 18, 2017 5
CDI: Life Sciences and Regenerative Medicine
2 Business Divisionso Life Sciences
o Cellular Therapeutics
Life Sciences Division serves 4 major market areaso Basic and Translational Sciences
o Safety Pharmacology & Toxicity
o Drug Discovery and Bioengineering
o Specialty Markets
Cellular Therapeutics Division has 2 focal areaso Internal cell therapy programs
o Ocular, Cardiac, Neurodegenerative, and Oncology
o Contract Development and Manufacturing partnerships
May 18, 2017 6
Life Science Research: Current Product Portfolio
iCell
Cardiomyocytes
iCell
Cardiac
Progenitor Cells
iCell
Neurons
iCell
DopaNeuronsiCell
Astrocytes
iCell
Motor NeuronsiCell
GlutaNeurons
iCell
HepatocytesiCell
Macrophages
iCell
Hepatoblasts
New Products
in development:
iCell RPEs
Early access availability
iCell
HPCs
iCell
Endothelial Cells
iCell
Skeletal MyoblastsMSCs
May 18, 2017 7
iCell Hepatocytes 2.0
Key Hepatocyte Characteristics
MorphologyAdherent monolayer; polarized phenotype; functional bile
canalicular network; polynucleation
Molecular
Markers
Albumin; α-1-antitrypsin; HNF family transcription factors
Intrinsic
Metabolism
Glycogen storage; lipid metabolism; insulin
responsiveness; urea synthesis
Phase I & II
Metabolism
- CYP3A4, 2C9, 2C19, 2D6, 1A2, 2B6, 2C8
- UGT, ST, GSTα activity
Transporter
Function
Transport via uptake (e.g. OATP, NTCP) and efflux
transporters (e.g. MDR-1/P-gp, BCRP, BSEP, MRP2)
Morphology, Transporter Function, Glycogen Storage
Alpha-1-antitrypsin
Albumin
50 µMPAS
Maturity Marker Expression
CDFDA
Day 14
CYP activities in the range of a random PHH donor panel
Activity prolonged over 7 days
CYP ActivityMetabolite Formation (LC/MS)
iCell Hepatocytes 2.0Morphological Characteristics
Adherent monolayer
Round nucleus
Distinct nucleoli
High cyto/nuclear ratio
Bi-nucleation (circles)
Bile canaliculi (arrows)
May 18, 2017 9
iCell HepatocytesHepatotoxicity Assessment (Cell Titer Glo) – 48hr exposure
iCell HCs PHH
Compound EC50 (AVG mM) EC50/CMax EC50 (mM) EC50/CMax
Acetaminophen 25 55.4 17.4 39.3
Troglitazone 0.045 2.0 0.033 1.5
Nefazodone 0.036 4.2 0.041 4.8
Theophylline >10 >100 >10 >100 Metabolism-Dependent toxicity
Key Points
Metabolism-dependent and
independent toxicity
detection
Results comparable to Gold
Standard
Lot to lot reproducibility
Benefit over other cell types
Metabolism competency
More functional than HepG2
Less variable than primary HCs
May 18, 2017 10
iCell Hepatocytes 2.0Long Term Culture Stability (on Collagen without any need for Matrigel)
Day 7 Day 14
Day 21 Day 28
May 18, 2017 11
iCell Hepatocytes Acute Vs Chronic Exposure
CompoundEC50 (uM)
2 Days 7 Days
Amiodarone 33.37 14.38
Minimal time - dependence Drastic temporal impact No temporal impact
Ability to test over sub-chronic exposure is a clear advantage over primary hepatocytes
May 18, 2017 12
iCell Hepatocytes – Now in 3D
iCell Hepatocytes 2.0 Are Capable of Forming 3D Spheroids
(A) An interconnected, confluent 2D culture
(B) 3D spheroids are generated within 1 – 2 days from pre-plated cells
Lower cell count and thus more cost-effective
May 18, 2017 13
0
500
1000
1500
2000
2500
3000
DIV 2 DIV 5 DIV 7 /Day 2
DIV 9 /Day 4
DIV 14 /Day 9
DIV 16 /Day 11
DIV 19 /Day 14
No
rmal
ize
d C
YP
3A
4 a
ctiv
ity
(RLU
/10
00
cel
ls/h
ou
r)
2D Culture: Days in vitro (DIV) or Spheroid culture: Day #
2D
3D
0
200
400
600
800
1000
1200
1400
1600
1800
DIV 2 DIV 5 DIV 7 /Day 2
DIV 9 /Day 4
DIV 16 /Day 11
DIV 19 /Day 14
DIV 21 /Day 16
No
rmal
ize
d U
rea
(pm
ole
s/h
ou
r/1
00
0 c
ells
)
2D Culture: Days in vitro (DIV) or Spheroid Culture: Day #
2D3D
Urea Secretion CYP3A4 Activity
Liver Spheroids Display Higher Levels of Urea and CYP3A4 Activity than 2D Cultures
Urea secretion levels and basal CYP3A4 activity (P450-Glo assay) was assayed over time in 2D and 3D
cultures of iCell Hepatocytes 2.0.
iCell Hepatocytes 2.0 spheroids exhibit elevated function
May 18, 2017 14
Cytochrome P450 basal activities – 2D vs. 3D cultures
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Acti
vit
y (
pm
ol/m
in/1
06 c
ells)
CYP1A2
2D 3D
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Acti
vit
y (
pm
ol/m
in/1
06 c
ells)
CYP2B6
2D 3D
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
Acti
vit
y (
pm
ol/m
in/1
06 c
ells)
CYP3A4/5
2D 3D
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Acti
vit
y (
pm
ol/m
in/1
06 c
ells)
CYP2C9
2D 3D
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Acti
vit
y (
pm
ol/m
in/1
06 c
ells)
CYP2D6
2D 3D
Characterization of Cytochrome P450 basal activities. iCell Hepatocytes 2.0 were cultured in 2D and 3D (spheroid)
formats out to 13 days post-thaw. Basal cytochrome P450 enzyme activities of CYP1A2, CYP2B6, CYP2C9, CYP2D6 and
CYP3A4/5 were determined with probe substrates phenacetin, bupropion, diclofenac, dextromethorphan and midazolam,
respectively, and analyzed by liquid chromatography paired with mass spectrometry (LC-MS/MS).
iCell Hepatocytes 2.0
May 18, 2017 15
CYP1A2
Hep
atocy
tes
1
2D C
ulture
3D C
ulture
0
2
4
6
8
10
12
14
16
18
20
(iCell Hepatocytes 2.0)
Acti
vit
y (
pm
ol/m
in/1
06 c
ell
s)
CYP2B6
Hep
atocy
tes
1
2D C
ulture
3D C
ulture
0123456789
101112
(iCell Hepatocytes 2.0)A
cti
vit
y (
pm
ol/m
in/1
06 c
ell
s)
CYP3A4/5
Hep
atocy
tes
1
2D C
ulture
3D C
ulture
0
5
10
15
20
25
30
35
40
(iCell Hepatocytes 2.0)
Acti
vit
y (
pm
ol/m
in/1
06 c
ell
s)
Characterization of phase I enzyme activity. iCell Hepatocytes 2.0 were cultured in 2D and 3D (spheroid) formats out to
13 days post-thaw. Basal cytochrome P450 enzyme activities of CYP1A2, CYP2B6 and CYP3A4/5 were determined with
probe substrates phenacetin, bupropion and midazolam, respectively, and analyzed by liquid chromatography paired with
mass spectrometry (LC-MS/MS). Example data from primary human hepatocytes (sandwich cultured for 5 days) is shown
for comparison purposes (data courtesy of QPS Hepatic Biosciences).
Phase I enzyme activity – Primary cells vs. iCell Hepatocytes 2.0
May 18, 2017 16
CYP3A4/5
Hep
atocy
tes
1
3D C
ulture
0
5
10
15
20
25
30
35
40
(iCell Hepatocytes 2.0)
Acti
vit
y (
pm
ol/m
in/1
06 c
ell
s)
Characterization of phase I enzyme activity.
iCell Hepatocytes 2.0, in 3D (spheroid) format
- CYP3A4/5 determined with;
- midazolam and analyzed by LC-MS (black
square)
- extrapolated from CYP3A4 P450-Glo data for 4
additional lots (blue triangles).
Primary hepatocyte data across different lots under
sandwich culture (data courtesy of QPS Hepatic
Biosciences).
3D iCell Hepatocytes yield consistent results
May 18, 2017 17
iCell Hepatocytes 2.0
Metabolism based toxicity testing
Extended culture time
- Enables subacute / chronic dosing
3D cultures
- Enhanced function
- Enhanced CYP activity
- Reproducible
CYP3A4/5
Hep
atocy
tes
1
3D C
ulture
0
5
10
15
20
25
30
35
40
(iCell Hepatocytes 2.0)
Acti
vit
y (
pm
ol/m
in/1
06 c
ell
s)
May 18, 2017 18
80K Cells/well80K Cells/well
High viability with bipolar or multipolar neurite outgrowths within days of plating
iCell GlutaNeuronsCharacterization
May 18, 2017 19
Characterization of iCell GlutaNeuronsPurity and Identity
Nestin
MA
P2
Nestin
TU
J
iCell GlutaNeurons are > 95% neuronal purity with ≥70%
Glutamatergic identity
Neuronal Identity Subtype Identity
May 18, 2017 20
Characterization of iCell GlutaNeuronsElectrophysiology on a Multi-Electrode Array System
iCell GlutaNeurons fire spontaneous action potentials
and form neural networks that burst synchronously
Single well
Individual
Electrodes
Periods of synchronized ‘firing’ across the well
May 18, 2017 21
iCell GlutaNeurons CDI exampleRobust Activity
48-well Plate (5X8: no data from the bottom row)
‘Velocity Graphs’
Raste
r P
lot
Raste
r P
lot
Velo
cit
y
Gra
ph
Ve
loc
ity
Gra
ph
= all-points histogram
binned @ 500 msec
May 18, 2017 22
All-points Histograms Graphs
*Note ALL wells are producing synchronous,
highly reproducible bursting phenotypes,
with very little noise between bursts and very
low CV IBIs
iCell GlutaNeurons: EPA Pre-drug dataRobust Activity
Key point: All wells showing synchronous activity
May 18, 2017 23
iCell GlutaNeurons & iCell AstrocytesBaseline data
Good Synchrony with or without iCell Astrocytes
May 18, 2017 24
iCell GlutaneuronsMonitoring electrical activity
Intercellular
(synaptic)
Activity
Intracellular
(ion channel)
Activity
iCell GlutaNeurons can be
used as a model to assess
changes in ion channel,
synaptic, and network activity
May 18, 2017 25
-200 -150 -100 -50 50 100 150 200
-200
-100
100
200
R2 = 0.76
Post WashoutPTX
PTX
D22
D200
D1800
-100
-50
0
50
100
150
200
250
300
mnFRs ActEsNA BPM
NA ConnchanB
chanBspkchanBDur
nBnBurstPerc
nBurstDurnElect
Synchrony
-100
100
300% Change
from Baseline
SpikesNetwork
‘Velocity’
Network‘ISI’
Channel‘Poisson’
SynchronyIndex
% C
ha
ng
e f
rom
Baselin
e
Network Burst
Frequency (BPM)
Poisson Burst
Intensity (Hz)
Post WashoutPTX (5 Minutes)
Vehicle66.7 33.3 16.7 8.3 4.1 2.2 1.11 H
ou
r P
ost
GB
Z (
µM
)
iCell GlutaNeurons: Seizurogenic Assay
May 18, 2017 26
iCell GlutaNeuronsExtended Pharmacology
iCell GlutaNeurons
Excitatory neuronal population
Seizurogenic model
Tested across chemical space
Utilized across multiple sites
Under evaluation within HESI
Translational Biomarkers of Neurotoxicity
May 18, 2017 27
iCell Neurons - Neurite Outgrowth24 Hour Outgrowth
The value lies in the ability to recreate several stages of development
and behavior and interrogate with multiple platforms.
May 18, 2017 28
Neurite Outgrowth and Enhancement HTSSherman et al., 2014 ISSCR Poster
Screened reference set of approximately 5,200 compounds
Identified compounds known to regulate neurite outgrowth
Identified 4 neurite outgrowth and 3 retraction additional hits not
predicted from current literature
Validates use for identifying changes in neurite morphology
May 18, 2017 29
Developmental Neurotoxicity ScreeningMultiplex Assay
Screened a diverse set of 80 chemicals for
potential DNT using HT HCI
Multiplexed endpoint assays: Neurite
outgrowth (NOG); cell viability; and
mitochondrial membrane potential (MMP)
Identified hits: 16 chemicals that had an
effect on NOG independent of cytotoxicity;
39 decreased MMP; and 9 were active in
both assays
Novel and improved methodologies over
animal assessment for DNT testing;
enables more rapid screening and
prioritization of potential neurotoxicants
May 18, 2017 30
Chemotherapeutic-induced NeuropathiesConsistent Batch Production Enables Phenotype Assays
Stem Cell Res, 2016
Different batches of iCell Neurons derived from a single iPSC clone showed:
• No significant differences in transcriptomic profiling
• Consistency in both gene expression and cytosine modifications; changed with
neurite growth as expected
• No significant differences in sensitivities to paclitaxel, vincristine, and cisplatin
May 18, 2017 31
Application of iPSC Neurons Chemotherapy induced Peripheral Neuropathy (CIPN)
Chemotherapeutics affect neurite outgrowth of iPSC neurons
Also have patient specific iPSC-based data
May 18, 2017 32
iCell Neurons
Show active neurite outgrowth
HCI procedures for neurite morphology quantification
Used successfully as a model for
Chemotherapy-induced Neuropathy
May 18, 2017 33
iCell Cardiomyocytes and iCell Cardiomyocytes2
Overview
Toxicity Testing Disease modeling /Target ID / Screening
Regenerative Medicine
Functional and Structural Toxicity Greater predictivity MOA Identification
Cardiac Patch / Catheter Delivery
C. Scott Tox Sci 2014
Guo 2011, 2013
HypertrophyDilated CardiomyopathyDiabetic CardiomyopathyIschemia/reperfusion
Regulatory Interactions >95% pure
Normal human biology
Predictive human reagent
Gold Standard
~100 publications
> 90% Top Pharma
Used by International
Regulatory Agencies
Human Cardiomyocytes
May 18, 2017 34
iCell Cardiomyocytes; Contextual RelevanceFunctional and Structural toxicity
Cell Signaling• Ca2+ signaling (EC coupling)
• Biochemical
Contextual relevance should enable both
functional and structural toxicity testing
Viability
Lipid accumulation
Mitochondrial function
Oxidative stress
Bioenergetics
etc…..
Structural Toxicity
1o effect is on general cellular processes
Electrical
Ion channels, Action Potentials, GPCRs
Functional Toxicity - 1o effect is on electrical/mechanical function
MechanicalContractility
May 18, 2017 35
iPSC within ½ log of rabbit wedge, in
some cases more sensitive
“MEA assays using iPSC-CMs offer a
reliable, cost effective, and surrogate to
preclinical in vitro testing, in addition to the
3Rs (refine, reduce, and replace animals in
research) benefit”Harris et al, Toxicol Sci 2013
Direct MEA readouts
iCell-Cs have been shown to provide
enhanced predictivity
iCell Cardiomyocytes Electrophysiologyshow predictive pharmacology
• >120 compounds
• ~equal positives and negatives
• beat rate, atypical beats, irregularity
> 90% -- QT prolongation> 80% -- Proarrhythmia
Guo et al., 2013
Guo et al., 2011
Also below for examples of Ca2+ surrogate readouts;
Lu et al., 2015; Pfeiffer etl al, 2016, Zeng et al., 2016;
Impedance surrogate readouts
May 18, 2017 36
iCell Cardiomyocytes Electrophysiologyshow reproducible results
Cross-site Baseline Parameters Cross-site Drug Responses
Amp
r2=0.84
BP
r2=0.88 FPDc
r2=0.91
Cross-site (2) study results from Phase II CiPA
iCell Cardiomyocytes2 and Maestro Apex Robot Platform
Baseline properties
Drug response (28 blinded compounds)
SOT 2017, Abstract 3346
May 18, 2017 37
iCell Cardiomyocytes Electrophysiologyshow reproducible results
Kitaguchi et al., J Pharm and Tox Methods, 2016
See also: Nozaki et al., Regul Toxicol Pharmacol, 2016
Nakamura et al., J Pharmacol Sci, 2014
Baseline properties across 10 facilities Drug response across 10 facilities
10 site study from CSA-Hi
iCell Cardiomyocytes and
MEA readout
Baseline properties
Drug response
Excellent electrophysiological cross site reproducibility
May 18, 2017 38
Dog cardiomyocytes
iCell Cardiomyocytes
Parameter IonOptix1 FLIPR2Impedance3
sensitivity 83% 77% 90%
specificity 84% 70% 74%
accuracy 82% 74% 84%
pos predict 90% 79% 85%
neg predict 76% 67% 82%
Comparisons between IonOptix-based measurements of dog cardiomyocytes (gold standard) to
Ca2+ and impedance-based measurements of iCell Cardiomyocytes (higher throughput)
1 AR Harmer Tox App Pharm (2012)2, A. Pointon Tox Sci (2014)3, C. Scott Tox. Sci (2014)C. Scott Tox Sci 2014
Measuring ContractilityComparison to Gold Standard
iCell Cardiomyocytes
• Show potency correlation with gold standard model
• Demonstrate good to excellent assay validation parameters
• provide a predictive surrogate model for measuring contractility
May 18, 2017 39
Implementing iCell Cardiomyocytes in toxicity testing cascade
Structural Toxicity
Cell injury
Cell death
Functional Toxicity
Proarrhythmia
Ca2+ handling
Contractility
Primary screen identifies a problem (or lack thereof)
Secondary investigations identify mechanism
Subsequent primary screens can be performed on med chem series
Peters et al., (2015) Cardiovasc Toxicol
May 18, 2017 40
KI-induced CardiotoxicityDeconvoluting the problem
FDA approved SMKI show cardiac liabilities
• Preclinical assays were insufficient
• Toxicities arose in late development / clinic
• Difficult to ascribe mechanism
Prediction hindered by :
• Highly conserved site of action-ATP-binding pocket
(on vs off target effects)
• Multiple effects on overlapping endpoints
Model can:
• Determine on-target vs off-target KI toxicity (MARK vs Chk KI)
• Identify KI-related toxicity with p<0.05
(>160 cmpds via Ambit and AZ-proprietary datasets)M. Peters CDI UGM 2014
Lamore et al., 2017
iCell Cardiomyocytes provide a predictive
tool for detecting KI toxicity
See also Cohen et al, 2013, Doherty 2013, Talbert 2014, Peters et al, 2015
S. Lamore SOT 2014
Cellular impedance assays with iCell
CMs can predict KI toxicity
Altered beat
phenotype indicates
upstream interaction
May 18, 2017 41
Case study: Off Target ToxicityGene Therapy Targeting MAGE A3 – Toxicity due to Titin-cross reactivity
•Modified T cell to increase affinity to MAGE 3A receptor, a
putative tumor antigen
• Phase I trial – 2 patients died of cardiogenic shock and
fever• Ventricular myofiber loss with infiltrate
• MAGE A3 not expressed on heart samples
• No toxicity in preclinical toxicity studies
•Bioinformatic modeling to detect - off target recognition of
titin, a protein that is a component of striated muscle•not expressed in static primaries
•only expressed in beating cells
•T cells expressing the affinity-enhanced TCR but not wild
type were toxic to iCell Cardiomyocytes
• Primary points•Affinity enhancing T cells may create unintended targets
…specificity testing to determine clinical suitability should
incorporate more biologically relevant culture systems
Cameron BJ, et al ; (2013) Sci Transl Med
Linette GP, et al ; (2013) Blood
May 18, 2017 42
iCell Cardiomyoytes / Cardiomyocytes2
Recapitulate native behavior
Predictive for proarrhythmia and altered contractility
Useful model for predicting adverse effects of small molecule and biologics-based oncology therapy
May 18, 2017 43
CDI iPSC Enabled Models:Existing and in Development (not exhaustive)
Cardiac Hepatic Neural
Arrhythmia HC Toxicity Seizurogenesis
Contractility 2D and 3D Excitotoxicity
Ca2+ handling CYP / Transporters Neurite outgrowth
Structural Toxicity NASH Synaptogenesis
Hypertrophy Malaria Alzheimer's (a-B, tau)
DCM HCV / HBV PD
Diabetes Diabetes ALS
Epilepsy
May 18, 2017 45