Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat!...
Transcript of Advances in Safety Pharmacology: Utilizing iPSC-derived ...The Heart Beat: A Remarkable Feat!...
Advances in Safety Pharmacology: Utilizing
iPSC-derived cardiomyocytes in early stage
safety pharmacology investigations
Blake Anson, PhD.
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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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Safety Pharmacology Key functional endpoints
Current common in-vitro models include cell lines and non-human cellular
preparations.
From M. Pugsley
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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.
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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
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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
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Why Human? : Very Different Ionic Mechanisms
Between Large Mammals and Rodents
Salama and London, J Physiol. 578:43-53, 2007
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Human Mouse
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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
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
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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
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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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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
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iPSC-cardiomyocytes utilize mitochondrial oxidative phosphorylation
Human iPSC Cardiomyocytes Metabolic Characterization
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Adapted from Rana et al., 2012
iPSC-cardiomyocytes utilize non-glycolytic substrates
iPSC-cardiomyocyte mitochondria are functional
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Human iPSC-Cardiomyocytes Contractility / Ca2+ handling Characterization
Determined by edge detection
Ca2+ Transients
Contraction
Sunny Z. Sun
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Human iPSC-cardiomyocytes Depolarizing Currents
Ma, et al, Am. J. Physiol., 2011
INa
ICa-L
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Human iPSC-cardiomyocytes Repolarizing Currents
Ma, et al, Am. J. Physiol., 2011
IKr
IKs
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Ifunny
IK1 Ito
iPSC-cardiomyocytes Other Currents
Ma, et al, Am. J. Physiol., 2011
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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
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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
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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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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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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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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
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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)
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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
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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
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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
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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
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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
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Human iPSC Cardiomyocytes Functional Utility - Contractility
Ca2+ Transients
Contraction
Sunny Z. Sun
Puppala et al, 2012
Determined by edge detection
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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
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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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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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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
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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
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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
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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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Example 1
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Example 1
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Example 2
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Example 2
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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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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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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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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