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Advances in Safety Pharmacology: Utilizing iPSC-derived cardiomyocytes in early stage safety pharmacology investigations Blake Anson, PhD.
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Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

May 30, 2020

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Page 1: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

Advances in Safety Pharmacology: Utilizing

iPSC-derived cardiomyocytes in early stage

safety pharmacology investigations

Blake Anson, PhD.

Page 2: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

2

Presentation Outline

I. Safety Pharmacology and in-vitro models

Need for human cardiac model

Potential for stem cell technology to provide a solution

II. Functional characterization of stem cell derived

cardiomyocytes

Genomic, protein, metabolic, electrophysiological

III. Methods for interrogating cardiomyocyte function

Electrophysiology, Ca2+ handling, Contractility

Direct and Indirect (screening) methodologies

IV. Case studies

Retrospective Analysis

V. Summary

Mechanistic and Phenotypic Approaches

Page 3: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

3

Safety Pharmacology Key functional endpoints

Current common in-vitro models include cell lines and non-human cellular

preparations.

From M. Pugsley

Page 4: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

4

4

4

The Heart Beat: A Remarkable Feat!

• Your heart is an electrically driven pump.

• It usually beats 60-80 times a minute,

or about 100,000 times a day,

or about 35 million times a year,

or about 3 billion times in a normal life span.

• If the normal pumping rhythm is severely

disrupted for more than a few minutes,

irreversible multi-organ damage and death

occur.

Page 5: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

5

5

5

iPSC-derived cardiomyocytes – Utility Cardiotoxicity: Two basic mechanisms

Drug induced Arrhythmias

Biochemical induced events

o Cell Permeability o Cell energetics o Oxidative stress o Mitochondrial dysfunction o Necrosis o Apoptosis

The two toxicities can be species specific and may not be causal or linked

The ideal test system is species specific and biologically relevant.

Torsades de Pointes

(Hismanal®) Vorperian et al, JACC, 15:1556-1561,

1996.

Human iPSC-derived cardiomyocytes

Page 6: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

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6

6

Why Human? Mammalian Heart Variability in Ventricular

Action Potential Duration

Guo et al, Heart Rhythm. 5:271-279, 2008

• Larger mammals have slower rates

• Longer APD

• Greater endo to epi dispersion of repolarization

Page 7: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

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7

7

Why Human? : Very Different Ionic Mechanisms

Between Large Mammals and Rodents

Salama and London, J Physiol. 578:43-53, 2007

P P

Human Mouse

Page 8: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

8 8

Human Stem Cell Derived Cardiomyocytes

Human cardiomyocytes

• ES or iPSC source material

• Recapitulate cardiac cellular behavior

• Unlimited Quantity

• High Purity

• Relevant Biology

• Broad End-use Platform Utility

Page 9: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

EMBRYONIC STEM CELLS

INDUCED PLURIPOTENT STEM CELLS

Source: CDI website; www.stemcelltechniques.blogspot.com

NEURONAL CELLS

HEMATOPOIETIC CELLS

CARDIOMYOCYTES

RENAL CELLS

HEPATOCYTES

Human Tissue Cells iPS Cell vs. ESC Origin

Page 10: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

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10

10

Stem Cell-Derived Models - Requirements Quality, Quantity, Purity

Cell

Pu

rity

Days in Culture

Target Cell (non proliferating)

Non-Target Cell (proliferating)

Key Characteristics for iPSC use in Safety Pharmacology

Purity

Highly pure cells yield accurate results

Quantity

Must be able to support large scale investigations

Quality Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype

Page 11: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

11

11

11 11

Presentation Outline

I. Safety Pharmacology and in-vitro models

Need for human cardiac model

Potential for stem cell technology to provide a solution

II. Functional characterization of stem cell derived cardiomyocytes

Genomic, protein, metabolic, electrophysiological

III. Methods for interrogating cardiomyocyte function

Electrophysiology, Ca2+ handling, Contractility

Direct and Indirect (screening) methodologies

IV. Case studies

Retrospective Analysis

V. Summary

Mechanistic and Phenotypic Approaches

Page 12: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

12 12

hiPSC-derived Cardiomyocytes Genomic and Transcript Characterization

~200 Cardiac genes were

identified from the Novartis GNF

expression atlas

• Expression levels of these

genes were obtained from

adult human heart mRNA

library (ambion)

• Expression levels of these

genes were obtained from pure

populations of iPSC-

cardiomyocytes over a 3

month period (d028 to d120)

• Relative levels compared with

each other

Comparative Analyses – whole genome profiling

Purified iPSC-Cardiomyocytes (iPSC-cardiomyocytes) show a stable cardiac

expression profile that matches adult human tissue

Adapted from Babiarz et al., 2011

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Human iPSC Cardiomyocytes Protein Expression

From Kattman et al., 2011

Page 14: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

14

iPSC-cardiomyocytes utilize mitochondrial oxidative phosphorylation

Human iPSC Cardiomyocytes Metabolic Characterization

14

Adapted from Rana et al., 2012

iPSC-cardiomyocytes utilize non-glycolytic substrates

iPSC-cardiomyocyte mitochondria are functional

Page 15: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

15 15

Human iPSC-Cardiomyocytes Contractility / Ca2+ handling Characterization

Determined by edge detection

Ca2+ Transients

Contraction

Sunny Z. Sun

Page 16: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

16 16

Human iPSC-cardiomyocytes Depolarizing Currents

Ma, et al, Am. J. Physiol., 2011

INa

ICa-L

Page 17: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

17 17

Human iPSC-cardiomyocytes Repolarizing Currents

Ma, et al, Am. J. Physiol., 2011

IKr

IKs

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18 18

Ifunny

IK1 Ito

iPSC-cardiomyocytes Other Currents

Ma, et al, Am. J. Physiol., 2011

Page 19: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

19 19

Spontaneous Action Potential Beating Rate (MEA):

Gai – m2

Carbachol

Gas – b1

Isoproterenol

Gaq – a1

Phenylephrine

Control Drug

Concentration (mM)

Fre

qu

en

cy

iPSC-cardiomyocytes GPCR Pathways

iPSC Cardiomyocytes have the appropriate GPCR pathways

Page 20: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

20 20

Stem Cell -Cardiomyocytes Action Potentials

Ma, et al, Am. J. Physiol., 2011

Peng et al., 2010

iPSC-CMS

hES-CMS

Stem cell derived CMs exhibit cardiac like action potentials

Page 21: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

21 21 21

Presentation Outline

I. Safety Pharmacology and in-vitro models

Need for human cardiac model

Potential for stem cell technology to provide a solution

II. Functional characterization of stem cell derived cardiomyocytes

Genomic, protein, metabolic, electrophysiological

III. Methods for interrogating cardiomyocyte function

Electrophysiology, Ca2+ handling, Contractility

Direct and Indirect (screening) methodologies

IV. Case studies

Retrospective Analysis

V. Summary

Mechanistic and Phenotypic Approaches

Page 22: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

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22

22

The Cardiac Action Potential

Schram et al. Circ Res 2002

Basic functional electrical unit

Drives contraction by initiating increases in intracellular Ca2+ levels

How are these processes interrogated in-vitro?

Fares and Howlett Proc. Aus. Physi. Soc. (2009)

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iPSC Cardiomyocytes Safety Pharmacology - APD Assay

iPSC-Cardiomyocytes show expected pharmacology

INa Block – Decreased dv/dt

ICa Block – shortened APD

IKr Block – prolonged APD

EADGeneration

% of control Dose Peak MDP APD10 APD50 APD90 dV/dt

TTX

N=5

1μM 103.8±3.0 100.3±0.9 107.1±2.5 103.1±1.9 101.7±2.0 76.7±7.4

3μM 100.0±1.3 99.5±0.8 105.7±4.3 100.6±2.5 98.8±0.8 41.2±11.2*

10μM 99.0±2.7 97.6±1.1 108.8±6.6 98.2±4.3 96.4±3.7 16.7±1.8*

30μM 99.1±3.6 96.2±2.1 112.4±6.3 102.0±3.4 100.2±3.1 16.8±2.0*

E4031

N=5

3nM 99.0±1.0 99.8±0.6 94.8±3.4 95.5±1.8 98.7±2.0 98.9±4.4

10nM 100.5±1.2 98.3±0.8 92.2±2.8 100.5±1.6 112.8±2.8 98.2±5.9

30nM 99.3±1.0 97.4±1.2 90.1±3.0 109.1±3.7* 140.3±7.6* 90.1±7.6

100nM 101.1±1.4 94.2±2.6 83.0±10.2 113.4±3.9* 170.42±13.6* 69.4±17.1

Nifedipine

N=5

3nM 89.5±4.9 99.5±0.4 83.3±7.1* 84.6±2.4* 89.4±1.0* 83.9±14.5

10nM 82.2±9.0 98.7±0.8 65.0±12.3* 70.3±6.1* 78.4±4.4* 91.3±4.4

30nM 87.2±6.9 97.3±1.8 60.9±7.6* 65.7±3.0* 74.0±2.3* 84.8±11.2

100nM 73.6±12.2 96.6±2.0 31.7±11.1* 45.4±4.5* 58.2±5.4* 87.7±8.6

Ma, et al, Am. J. Physiol., 2011

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24

hESC Cardiomyocyte AP Pharmacology

Arrhythmic Triggers

Response Across Ion Channel Families

Quantifiable Responses

Peng et al., 2010 heSC-Cardiomyocytes show expected pharmacology

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25 25

Stem cell Cardiomyocytes recapitulate

pro-arrhythmia triggers EAD amplitude varies inversely with take off potential

Ma et al, AJP:H&C, 301:H2006+, 2011

iPSC CM

APs

Adult Canine

Purkinje Fiber

APs

January et al, Circ Res, 65:570+, 1988

Bay k 8644

EAD

Take off

potential

-2.28±0.11 mV/mV -1.87±0.26 mV/mV

Page 26: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

26

sfAPDc

0 10 30 100

200

300

*** ***

Nifedipine (nM)

0 3 10 30 100 200

300

400 *** *

*

E-4031 (nM) 0 3 10

100

200

300 **

Chromanol 293B (mM)

Field

Potential

Overlays

msec

-200

-100

0

100

Nifedipine

mV

control

10 nM

30 nM

-100

0

E-4031

mV

control

3 nM

10 nM

30 nM

100 nM

-100

0

100

Chromanol 293B

mV

control

10 mM

30 mM

Compounds induce expected effects

Technique

100 uV

500 ms

Waveforms 1 – 64 wells

at a time

Human iPSC Cardiomyocytes Organotypic Recordings MultiElectrode Array (MEA)

Page 27: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

27

iPSC Human Cardiomyocyte FPD Initiation

depends on well position within the plate

iPSC Human Cardiomyocyte Field Potentials

48-well MEA plate

ms

0

1

2

3

4

iPSC Conduction velocity

can be measured

Human iPSC Cardiomyocytes Multi-electrode Array – Measuring Conduction Velocity

Organotypic preparations provide additional/alternative endpoints

Drug-induced effects on conduction velocity

can be measured

Page 28: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

28 28

iPSC Cardiomyocytes – Utility Ca2+ Measurements

Calcium 5 Assay Kit (Molecular Devices)

Fluorescence intensity of Ca2+ transients from autonomously beating cells.

The entire experiment was finished in 100 seconds.

0 20 40 60 80 100100

200

300

400

Addcompound

Time, sec

RF

U

Beat rates before and after addition

of 0.3 mM epinephrine

Positive

Chronotropes:

Negative

Chronotropes:

Isoproterenol

Epinephrine

Dopamine

Doxasozine

Verapamil

PropranololConcentration, uM

1e-4 0.001 0.01 0.1 1 10 100

0

10

20

30

40

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2

Plot#5 (Doxazosin: Concentration vs MeanValue) 19 0.37 1.1e+16 -4.6e+06 0.976

Isoproterenol (Isoproterenol: Concentration vs Me... 18.2 1.25 0.00201 38.5 0.983

Plot#3 (Epinephrine: Concentration vs MeanValue) 17.4 0.576 0.00919 38.1 0.965

Plot#2 (Propanolol: Concentration vs MeanValue) 19.4 0.692 1.49 6.87 0.974

verapamil (Verapamil: Concentration vs MeanVal... 18.4 0.642 0.0112 -0.669 0.978

Dopamine (Dopamine: Concentration vs MeanVal... 17.5 0.673 0.339 36.2 0.987__________

Weighting: Fixed

Concentration, uM

1e-4 0.001 0.01 0.1 1 10 100

0

5

10

15

20

25

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2

Plot#1 (Astemisole: Concentration vs MeanValue) 13.7 29.4 0.0744 -1.25e-12 0.995

Plot#5 (TTX: Concentration vs MeanValue) 15.8 0.819 2.69 4.2 0.889

Plot#4 (Cisarpide: Concentration vs MeanValue) 13.4 2.23 0.0432 0.0999 0.98

Plot#2 (Pimoside: Concentration vs MeanValue) 14.5 85.9 0.292 3.64e-22 0.985

Plot#3 (Terfenadine: Concentration vs MeanValue) 13.7 3.25 0.455 0.0588 0.993__________

Weighting: Fixed

Be

ats

/min

384w format

Concentration, uM

0.001 0.01 0.1 1 10

0

10

20

30

40

50

60

70

80

90

100

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2

iso (Isoproterenol: Concentration vs MeanValue) 32.1 1.51 0.0075 87.3 0.998

doxa (doxazosin: Concentration vs MeanValue) 31.6 2.28 0.626 11.4 0.826

vera (verapamil: Concentration vs MeanValue) 28.9 1.03 0.0534 -0.214 0.983

epi (Epinephrine: Concentration vs MeanValue) 30.6 0.734 0.0155 83.5 0.999

dopamine (didoxine: Concentration vs MeanValue) 32.7 0.893 0.187 58.5 0.99__________

Weighting: Fixed

96w format

Be

ats

/min

Modified from Sirenko et al., 2013

Page 29: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

29 29

iPSC Cardiomyocytes – Utility Ca2+ Imaging

Calcium 5 Assay Kit (Molecular Devices)

Fluorescence intensity of Ca2+ transients from autonomously beating cells.

The entire experiment was finished in 100 seconds.

0 20 40 60 80 100100

200

300

400

Addcompound

Time, sec

RF

U

Beat rates before and after addition

of 0.3 mM epinephrine

Calcium 5 Response on FLIPR: Epinephrine High Dose (C06)

-20000

-10000

0

10000

20000

30000

40000

10.5 11 11.5 12 12.5 13 13.5

Time (sec)

Amplitude

& Time

Decay Time

Rise

Time

Peak Width

(FWHM)

New algorithms are being developed for analysis

Page 30: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

30 30

Human iPSC Cardiomyocytes Functional Utility - Contractility

Ca2+ Transients

Contraction

Sunny Z. Sun

Puppala et al, 2012

Determined by edge detection

Page 31: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

31 31

Human iPSC Cardiomyocytes Relevant Mitochondrial Toxicity Screens

Assess viability while inhibiting

mitochondrial function (rotenone, antimycin, and oligomycin)

Decreased viability is masked by glucose

Media matters! Context inappropriate media

can mask outcomes

Adapted from Rana et al., 2012

Page 32: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

32 32 32

Presentation Outline

I. Safety Pharmacology and in-vitro models

Need for human cardiac model

Potential for stem cell technology to provide a solution

II. Functional characterization of stem cell derived cardiomyocytes

Genomic, protein, metabolic, electrophysiological

III. Methods for interrogating cardiomyocyte function

Electrophysiology, Ca2+ handling, Contractility

Direct and Indirect (screening) methodologies

IV. Case studies

Retrospective Analysis

V. Summary

Mechanistic and Phenotypic Approaches

Page 33: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

33 33

iPSC Cardiomyocytes – Screening Ca2+ Imaging reflects electrical activity

Calcium 5 Assay Kit (Molecular Devices)

Fluorescence intensity of Ca2+ transients from autonomously beating cells.

The entire experiment was finished in 100 seconds.

0 20 40 60 80 100100

200

300

400

Addcompound

Time, sec

RF

U

Beat rates before and after addition

of 0.3 mM epinephrine

Calcium 5 Response on FLIPR: Epinephrine High Dose (C06)

-20000

-10000

0

10000

20000

30000

40000

10.5 11 11.5 12 12.5 13 13.5

Time (sec)

Calcium 5 Dye Response on FLIPR - Cisapride High Does (I12)

-10000

0

10000

20000

30000

40000

50000

60000

70000

13000 15000 17000 19000 21000 23000

Time (ms)

Width ~ 1 sec Width ~ 8 sec

Electrical activity at the membrane is reflected in the Ca2+ transient

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34 34

iPSC-cardiomyocytes – Screening FLIPR® Tetra System - Quantitation

K7 - Group:Cisapride_9 - Concentration:0.015242

Time (Seconds)

0 20 40 60 80 100

Rela

tive L

ight U

nits

300

400

I7 - Group:Cisapride_7 - Concentration:0.137174

Time (Seconds)

0 20 40 60 80 100

Rela

tive L

ight U

nits

200

300

400

Cisapride 15 min post addition

[Cisapride] (uM)

0.01 0.1 1 10 100

Peak C

ount (1

20 s

ec)

0

5

EC/IC50 = 0.1446

No Treatment 0.14 mM Cisapride

K7 - Group:Cisapride_9 - Concentration:0.015242

Time (Seconds)

0 20 40 60 80 100

Rela

tive L

ight U

nits

300

400

I7 - Group:Cisapride_7 - Concentration:0.137174

Time (Seconds)

0 20 40 60 80 100

Rela

tive L

ight U

nits

200

300

400

Cisapride 15 min post addition

[Cisapride] (uM)

0.01 0.1 1 10 100

Peak C

ount (1

20 s

ec)

0

5

EC/IC50 = 0.1446

No Treatment 0.14 mM Cisapride

Intracellular Ca2+ oscillations can be used to measure membrane ion channel activity In 96-384 well plates

Page 35: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

35

35

35

96-well E-eplate

A surrogate measurement platform for electrical activity, Ca2+ handling, contractility

Gold electrode

0.1

1

10

0 20 40 60 80 100 120 140 160 180

Time (hour)

Ce

ll In

de

x (

log

)

Incre

asin

g Im

ped

an

ce

Seconds

Slide modified from K. Kolaja

Human iPSC Cardiomyocytes Label-Free Monitoring xCelligence RTCA Cardio

Page 36: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

36

36

36

Arrhythmia Screening in 96-wells

iPSC-cardiomyocytes - Utility Label-Free Measurements – Arrhythmogenesis

PPS - Predicted ProArhythmia Score

More predictive than current models

Guo et al., 2011

Identifies False

Negatives

identifies False

Positives

Page 37: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

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37

37 37

Presentation Outline

I. Safety Pharmacology and in-vitro models

Need for human cardiac model

Potential for stem cell technology to provide a solution

II. Functional characterization of stem cell derived cardiomyocytes

Genomic, protein, metabolic, electrophysiological

III. Methods for interrogating cardiomyocyte function

Electrophysiology, Ca2+ handling, Contractility

Direct and Indirect (screening) methodologies

IV. Case studies

Retrospective Analysis

V. Summary

Mechanistic and Phenotypic Approaches

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38

Example 1

Page 39: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

39

Example 1

Page 40: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

40

Example 2

Page 41: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

41

Example 2

Page 42: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

42 42 42

Presentation Outline

I. Safety Pharmacology and in-vitro models

Need for human cardiac model

Potential for stem cell technology to provide a solution

II. Functional characterization of stem cell derived cardiomyocytes

Genomic, protein, metabolic, electrophysiological

III. Methods for interrogating cardiomyocyte function

Electrophysiology, Ca2+ handling, Contractility

Direct and Indirect (screening) methodologies

IV. Case studies

Retrospective Analysis

V. Summary

Mechanistic and Phenotypic Approaches

Page 43: Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat! •Your heart is an electrically driven pump. •It usually beats 60-80 times a minute,

43

Functional Endpoints

Example subset

Process Cell Biology “Wetware”

Endpoint Platform

Electrical

activity

Ion channels

GPCRs

AP perturbation

Chronotropic

changes

Patch Clamp,

MEA, FliPR, HCI,

Label-free

Ca2+

handling

RYR2

SERCA

Release and

uptake

FliPR, HCI, label-

free

Contractility Contractile

filaments

Inotropic changes HCI, Ionoptix,

label-free

Bioenergetics Fatty Acid

Oxidation

Lipid

Metabolism

Mitochondrial

function

ATP production

Oxygen

consumption,

mitochondrial

dyes

Mech

an

isti

c A

pp

roach

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44

Functional Endpoints

Example subset

Process Cell Biology “Wetware”

Endpoint Platform

Electrical

activity

Ion channels

GPCRs

AP perturbation

Chronotropic

changes

Patch Clamp,

MEA, FliPR, HCI,

Label-free

Ca2+

handling

RYR2

SERCA

Release and

uptake

FliPR, HCI, label-

free

Contractility Contractile

filaments

Inotropic changes HCI, Ionoptix,

label-free

Bioenergetics Fatty Acid

Oxidation

Lipid

Metabolism

Mitochondrial

function

ATP production

Oxygen

consumption,

mitochondrial

dyes

Phenotypic Approach Consistent

output

Surrogate

measurements

Mech

an

isti

c A

pp

roach

ad

vers

e

sig

nal

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45 45

Summary

Human cardiomyocytes

Provide an in-vitro recapitulation of native cellular biology and physiology

Can be used across a variety of interrogation platforms

Have, in some cases, shown greater utility than current models

Can be used for phenotypic and mechanistic screening investigations

Human Stem Cell Derived Cardiomyocytes

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Thank-you