Wh t M k C di tWhat Makes a Cardiomyocyte a Cardiomyocyte?

Kyle KolajaKyle Kolaja

March 14th 2013

What Makes a Cardiomyocyte a Cardiomyocyte?

Shouldn’t proliferateproliferate

Should beat

Should be fairly pure

Should look like and express genes like a heart

llcell

Should respond to cardioactive

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to cardioactivedrugs

Cardiomyocyte a Cardiomyocyte

Rationale from a toxicologist perspective

Two schools of thought Bits and pieces look right Cell-based system responds like a heart

Bits and Pieces Characterization Gene expression Gene expression Channel activity/function Purity and impact on endpoints

Cell-based systems Arrhythmia

H t h Hypertrophy Toxicity

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iCell Cardiomyocytes are more similar to Adult Human Heart Samples than Primary Cultures

CDI ll d28 d d120CDI cells d28 and d120 Primary Heart cultures

Troponin I = at background = ES cell

4Babiarz et al Stem Cells Dev. Jan 2012

iCell Cardiomyocytes are more similar to Adult Human Heart Samples than Primary Cultures

CDI cells d28 and d120 Primary Heart cultures

Troponin I = at background = ES cell

5Babiarz et al Stem Cells Dev. Jan 2012

Cardiomyocyte Manufacturing Process

CREATE &MAINTAIN DIFFERENTIATEEXPAND

Cardiomyocyte

FINSH & CRYOPRESERVE

y yProduction

• Quality• Quantity• Purity

Purity and Morphology

Differentiation with expression-based selection can yield >98% purity

Cryopreservation makes easier for everyone Cryopreservation makes easier for everyone Morphology with sarcomeres & striated myofibers

Confirmation of Cardiomyocyte Nature

iCell CardiomyocytesExpress major cardiac proteinsExhibit cardiac action potential wave formsFunctionally active cardiac channelsRespond as expected to channel blockers

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Ma et. al Am J Physiol Heart Circ Physiol. (2011) High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents

Impedance Detection of Cardiomyocyte Beating

96 ll E l t96-well E-eplate

Seconds

10

log)

1

Cel

l Ind

ex (

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0.10 20 40 60 80 100 120 140 160 180

Hours

Drug-induced changes in beat rateIndication of functional cardiac ion channels

xCELLigence: Impedance MEA: Electrical Field

Control

Na+ Channel Blocker

Pace-maker Current Blocker

Agonist of β-Adrenergic

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gReceptor

Does impedance detect the physical beating or the electrical changes?

Pre-drug

Blebbistatin:Blocks myosin II in an actin-detached state 15 min MEA 15 minPre drug 15 min

0 µM

MEA 15 min

0.10

0 30

0.03

0.30

1.

3.

30

10

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In vitro Detection of Arrhythmias with Human iCell Cardiomyocytes

Pre drug Post drugPre-drug Post-drug

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In vitro Detection of Arrhythmias using Human iCell Cardiomyocytes

DMSO

E-4031 causes arrhythmia

E-4031

Nifedipine rescues E-4031-induced arrhythmia

DMSO + Nif

E-4031+ Nif

Misner et al Br JMisner et al Br J Pharmacol. 2012 Apr;165(8):2771-86.

12Functional Rescue of Drug-induced Arrhythmia

Human Cardiomyocytes (hCAR) AssayiPS cell-derived human cardiomyocytes

IB20= lowest tested concentration resulting in 20%

irregular beats

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hCAR Assay Validation:23 compounds with known in vivo effect

DrugIB20 (µM)

hERG QTClinical

arrhythmiaDofetilide 0.003 (+) (+) (+)

•12 Pro-arrhythmic• 11 Non-arrhythmic• IB20 30 uM

Ouabain 0.03 (‐) (‐) (+)Aconitine 0.03 (‐) (‐) (+)Cisapride 0.03 (+) (+) (+)E‐4031 0.03 (+) (+) (+)Astemizole 0.03 (+) (+) (+)

• One False Positive• No False Negatives

Terfenadine 0.3 (+) (+) (+)Flecainide 1 (+) (+) (+)Alfuzosin 1 (‐) (+) (‐)Thioridazine 3 (+) (+) (+)Quinidine 10 (+) (+) (+)E h i 30 ( ) ( ) ( )Erythromycin 30 (+) (+) (+)Sotalol 30 (+) (+) (+)Fluoxetine >30 (+) (+) (‐)Verapamil >30 (+) (±) (‐)Moxifloxacin >100 (+) (+) (+)

• IB20= lowest tested concentration resulting in 20% i l b

Amiodarone >100 (+) (+) (+)Ranolazine >100 (+) (+) (‐)Captopril >100 (‐) (‐) (‐)Rofecoxib >100 (‐) (‐) (‐)Amoxicillin >1000 (‐) (‐) (‐)

irregular beats Aspirin >1000 (‐) (‐) (‐)Nifedipine >3 (‐) (‐) (‐)

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Guo et al. Toxicol Sci. 2011 Sep;123(1):281-9.

Human Cardiomyocytes (hCAR) AssayNext Round of Validation

83 Compoundsp

~82% -- arrhy. prediction>90% -- QT prediction

30 Internal30 Internal Compounds

80% -- arrhy. prediction95% -- QT prediction

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Hallmarks of Cardiac Hypertrophy

• Increased cell size• Enhanced protein synthesisy• Higher organization of the sarcomere• Re-activation of the fetal gene program• Expression of B-type natriuretic protein (BNP)

NormalUntreated + PE

Kuwahara K et al. J Pharmacol Sci 2012;119:198-203

Cel

l Siz

e

Protein Synthesis BNP Expression

ctin

Reo

rg

DiseasedLister K et al. Cardiovasc Res 2006;70:555-565

Ac

Glenn D et al. Hypertension 2009;53:549-555

Hypertrophy Application Suite

qPCR• Measure activation of BNP gene

BNP (NPPB)

ELISA• Quantify levels of secreted BNP• Quantify levels of secreted BNP

Flow Cytometry• Detect BNP expression

High Content Analysis• Image BNP expression, Cell Size CellSize, Cell

Thaw and Plate iCell Cardiomyocytes

Induce with ET-1

Detect BNP

Coat Plate with Fibronectin Replace Medium

0 2 4 5

NHLBI Family Blood Pressure Program –GWAS now iCell Cardiomyocytes

• GWAS identification of associated SNPs with left Ventricular Hypertrophy• Phenotyping through cardiovascular phenotypes and risk factors• Family-based ascertainment

• NHLBI, Next Generation Genetic Association Studies, RFA-HL-11-066• CDI will make pure ventricular cardiomyocytes from 250 different patients• 100 most informative families derived from HyperGEN cohort• Induce hypertrophy phenotype in vitro, perform molecular analyses• Attempt to correlate GWAS findings with in vitro phenotype

CDI Grant Milestones

You are here

Grant Year 1 2 3 4 5

Calendar Year 2011 2012 2013 2014 2015 2016Year 2011 2012 2013 2014 2015 2016

Research Sample identification and Optimize parallel iPS and CM production

Grant Phases Development

Cell Production

Production Plan 100 130 70

donors donors donors25 donors completed or in progress

Example Pre and Post Selection Purity and YieldSample ID O1144

Start of Selection (day 12) End of Selection (day 16)

cTnT-33 0% T T 88 9%

CM yield (start of selection): 256M CM yield (end of selection): 280M

cTnT-33.0% cTnT-88.9%

QC Criteria at cryo (day 30)• >90% purity (cTnT+)p y ( )

• > 4.3 million viable cells per cryovial

• >50 million total yield (200M target)

• Mycoplasma negative

This will drift up to ~97% by day 30

y p g

• Identity screen (STR) match

• Observed beating rate

Studying the mechanism of Sunitinib (Sutent) mediated cardiotoxicity

Sutent multi-targeted (RTK) inhibitor for oncology g ( ) gy

indication Cardiac dysfunction and toxicity

congestive heart failure and left ventricular systolic g ydysfunction

Inhibits hERG and causes arrhythmia Previous work done mostly with NRVMs

Binds 150 kinases Inhibits proliferation/survival/angiogenesis

VEGFR1-3, PDGFR(α and β), c-Kit, FLT3, CSF1R, and RET

AMP Kinase – cellular gas gauge RSK – ribosomal s6 kinase -downstream of ERK

Aurora Kinase - cell prolif/, Chromatid seg., mitosis

Advancing the Understanding of Sutent Cardiotoxicity

Sutent potently cytotoxic in iCell CMs

Confirmed the inhibition of AMPK by sutent but Confirmed the inhibition of AMPK by sutent but minimal protective effect seen by AMPK agonists

Inhibit hERG, Ca++ cycling and NaV1.5 -> Inhibit hERG, Ca++ cycling and NaV1.5 > arrhythmia

RSK inhibitors not cardiotoxic iCell CMs ideal RSK inhibitor inhibits hERG and causes arrhythmia

Aurora Kinases

model to assess cardiotoxicity,

electrophysiology d t tilit

Multi-factorial combination of cytotoxicity, cardiac conduction abnormalities, hypoxia, pan-kinase inhibition leading to suppressed

and contractility effects in parallel

kinase inhibition leading to suppressed response/accommodation mechanisms

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Jaspamide Cardiotoxicity

Jaspamide (a cyclodepsipeptideisolated from the marine spongeisolated from the marine sponge Jaspis johnstoni ) a potential cancer therapeutic agent

Pulmonary edema and cardiac hemorrhage and congestion in preclincal tox species

iCell CMs ideal

Jaspamide (10 lM) inhibited Kv1.5 activity by 98.5%. And also inhibited

iCell CMs ideal model to assess cardiotoxicity,

electrophysiology Cav1.2, Cav3.2, and HCN2; but not hERG

p y gyand contractility effects in parallel

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Affects actin binding to cytoskeleton

Opportunity for iPS-derived Cardiomyocytes in Cardiac Research

Functional aspects of PSC-derived cardiomyocytes highlight an improved in vitro tool for studying cardiotoxicitycardiotoxicity Gene expression Channel activity/function Purity and impact on endpoints Rhythmic beating and response to treatment Doesn’t proliferate Doesn t proliferate Maintain in culture for prolonged periods

Example cell-based systems just beginning Arrhythmia and hypertrophy Mechanistic toxicity – regulatory filings

Continued validation through empirical testing24