ACEA Biosciences & FUJIFILM Cellular Dynamics, Inc.

Presentation

The Utility of Electrical Field Stimulation for Functional Maturation of hiPSC-CM & Assessment of Inotropic Compounds

March 11, 2019

T.K. Feaster, PhD

Outline of Presentation

1. FUJIFILM Cellular Dynamics, Inc. and iPSC Technology

2. ePacer and CardioECR

3. Positive Force-Frequency and Inotropic Response

4. Detection of outlier CiPA drugs

5. Summary

FUJIFILM Cellular Dynamics iPSC Technology

Applications Support

BasicScience

Healthy donor orPatient-derived

DrugDiscovery

CellTherapy

BRAIN & NERVOUS SYSTEMiCell Astrocytes

iCell DopaNeurons

iCell GABANeurons

iCell GlutaNeurons

iCell Motor Neurons

iCell Microglia

HEART & VASCULARiCell Cardiomyocytes

iCell Endothelial Cells

iCell Cardiac Progenitor Cells

LIVERiCell Hepatocytes 2.0

iCell Hepatoblasts

OCULARiCell Retinal Pigment Epithelial Cells

iCell Hematopoietic Progenitor Cells

iCell Macrophages

BLOOD & IMMUNE

iCell® Products: Unprecedented Access to Human Cells

iCell CardiomyocytesExcitation-Contraction Coupling

Electrical Activity Calcium Handling Mechanical Beating

iCell Cardiomyocytes are spontaneously beating cells that are ideal for interrogation of excitation-contraction coupling.

Xiulan Yang et al. Circulation Research. 2014

ACEA ePacer with CardioECR system

ePacer enables electrical stimulation in assay plate.

ePacer enables monitoring of cells during pacing, and optimization of pacing signal.

Built in function to graph Force-Frequency relationship

CardioECR system records both impedance and MEA signals.

Negligible cell loss

Experimental Design

Thaw and Plate Cells into E-Plate according

to App Protocol

Days in Culture 0 224h

iCMM media changeEvery 48 hours

ICPM to ICMM Perform FFR Test, Stop Pacing

Compound Addition

Record 30 Minutes

Spontaneous

Donor: 01434CaucasianFemaleAge Group:0 - 18

Donor: 11713CaucasianFemaleAge Group:30 - 40

7

Pace 1.0Hz(5 Days)

12

Pace 1.5 Hz(5 Days)

17

Pace 2.0Hz(5 Days)

iCMM media changeEvery 24 hours

iCellCardiomyocytes,11713

iCellCardiomyocytes2,01434

ePacer Representative Waveforms

1.0Hz

1.5Hz

2.0Hz

Unpaced Control PacedSpontaneous

Spontaneous

Spontaneous

Day 12

Day 17

Day 22

Pacing Induced Positive Force-Frequency Relationship in hiPSC-CMs

- Unpaced Control- Paced

iCell Cardiomyocytes,11713

iCell Cardiomyocytes2,01434

Unpaced Control

Unpaced Control

Paced

Paced

DIV 22

1.0 Hz0.5 Hz 2.0 Hz1.5 Hz

1.0 Hz0.5 Hz 2.0 Hz1.5 Hz

ePacer and Cardio Baseline DataBeat Rate Amplitude

Control

Paced

0.0

0.5

1.0

1.5***

Nor

mal

ized

Bea

t Rat

e

Control

Paced

0.0

0.5

1.0

1.5*

Nor

mal

ized

Bea

t Rat

e

Control

Paced

0.0

0.5

1.0

1.5***

Nor

mal

ized

Am

plitu

de

Control

Paced

0.0

0.5

1.0

1.5***

Nor

mal

ized

Am

plitu

de

iCellCardiomyocytes,

11713

iCellCardiomyocytes2,

01434

DIV 22

Inotropic Response in hiPSC-CMs

-11 -10 -9 -8 -7 -60

20

40

60

80

Log Isoproterenol [M]

Bea

t R

ate

(s)

~ EC50 27 nM

-11 -10 -9 -8 -7 -60

20

40

60

80

Log Nifedipine [M]

Bea

t R

ate

(s)

-11 -10 -9 -8 -7 -60.00

0.05

0.10

0.15

0.20

0.25

Log Isoproterenol [M]

Am

plitu

de (

CI)

-11 -10 -9 -8 -7 -60.00

0.05

0.10

0.15

0.20

0.25

Log Nifedipine [M]

Am

plitu

de (

CI)

iCell Cardiomyocytes, 11713

~ EC50 17 nM

~ EC50 70 nM

~ EC50 7 nM

~ EC50 13 nM

~ EC50 12 nM

~ EC50 10 nM

~ EC50 10 nM

Unpaced ControlPaced

-11 -10 -9 -8 -7 -60

20

40

60

80

Log Isoproterenol [M]

Bea

t R

ate

(s)

-11 -10 -9 -8 -7 -60

20

40

60

80

Log Nifedipine [M]

Bea

t R

ate

(s)

-11 -10 -9 -8 -7 -60.00

0.05

0.10

0.15

0.20

0.25

Log Isoproterenol [M]

Am

plitu

de (

CI)

-11 -10 -9 -8 -7 -60.00

0.05

0.10

0.15

0.20

0.25

Log Nifedipine [M]

Am

plitu

de (

CI)

iCell Cardiomyocytes2,01434

~ EC50 41 nM

~ EC50 20 nM

~ EC50 NA

~ EC50 24 nM

~ EC50 13 nM

~ EC50 15 nM

~ EC50 10 nM

~ EC50 10 nM

DIV 22

Multiplexing with the E-Plate Cardio VIEW 96

Paced hiPSC-CMs Display Isoproterenol Induced Increased

Calcium Transient Amplitude Viewing Window Enables Immunofluorescent Assessment

Vehicle Isoproterenol [0.1 µM]

UnpacedControl

Paced

Green = Cardiac Troponin T (cTnT)Blue = DAPI

Electrodes

Detecting Outlier CiPA Drugs

https://www.ncbi.nlm.nih.gov/pubmed/30257217

hiPSC-CMs improve torsades de pointes arrhythmia prediction. Proarrhythmic detection with greater than 87% predictivity. Consistent data across multiple laboratories and platforms.

Outlier Drugs:Bepridil, Ranolazine,

Metoprolol, Mexiletine

Low TdP Risk Compound Respond Appropriately in Paced hiPSC-CMs

Vehicle

ECR

Mexiletine [3.0 µM] Metoprolol [33 µM] Bepridil [0.3 µM]

Impedance

Low TdP Risk High TdP Risk

DIV 22

Cmax [0.03 µM]Cmax [1.8 µM]Cmax [2.5 µM]Cmax [1.9 µM]

Ranolazine [11 µM]

CiPA [10 µM] CiPA [100 µM]CiPA [100 µM] CiPA [100 µM]

Cmax N.A.

CiPA N.A.

0.03 0.1 0.3 1 3 11 33 100

0

10

20

30

40

50

[M]

% Ir

regu

larit

y

0.03 0.1 0.3 1 3 11 33 100

0

10

20

30

40

50

[M]

% Ir

regu

larit

y

0.03 0.1 0.3 1 3 11 33 100

0

10

20

30

40

50

[M]

% Ir

regu

larit

y

0.03 0.1 0.3 1 3 11 33 100

0

10

20

30

40

50

[M]

% Ir

regu

larit

y

0.03 0.1 0.3 1 3 11 33 100

0

10

20

30

40

50

[M]

% Ir

regu

larit

y

Q Q QQ Q Q Q Q Q Q

Q = Quiescence

Q

Summary

•Positive force-frequency relationship induced by pacing hiPSC-CMs with the ePacer

•Improved isoproterenol positive inotropic response in paced hiPSC-CMs

•Detection of low TdP Risk CiPA outlier drugs after pacing

•Pacing results in altered baseline beat rate and reduced impedance amplitude

Acknowledgements

FUJIFILM Cellular Dynamics•Research and Development

• Nathan Meyer• Tom Burke

•Applications Development• Michael Hancock

•Product Development• Simon Hilcove• Genie Jones

ACEA•Xiaoyu Zhang•Jeff Xue•Yama Abassi

Thank you