Human induced pluripotent stem cell derived
cardiomyocytes are a more relevant model for
assessing drug-induced effects on mitochondrial
function than H9C2 cells
16 January 2013
SLAS2013
Presentation Outline
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Stem cell technology
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iCell Cardiomyocytes (hiPSC-derived CMs)

Genomic Characterization

Functional Characterization

Bioenergetic Characterization

Model Comparison
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Stem Cell Sources
iPS Cell vs. ESC Origin
INDUCED PLURIPOTENT STEM CELLS
NEURONAL
CELLS
HEMATOPOIETIC
CELLS
CARDIOMYOCYTES
RENAL CELLS
HEPATOCYTES
EMBRYONIC STEM CELLS
Source: CDI website; www.stemcelltechniques.blogspot.com
iPS Cell Technology
Revolutionary Access to Human Biology
Edit any gene
in the genome
Mastery of reprogramming,
differentiation, and
engineering has the potential
to revolutionize biological
sciences
208 Cell Types
Differentiation
Differentiate into
all 208 cell types in
the human body
6 Billion People
Reprogramming
Represent any
individual
genotype
4
Company Overview

Cellular Dynamics International (CDI) is the world’s largest producer of human iPS cells
and iPS cell-derived cell types

Headquartered in Madison, WI

Currently employs >100 total staff (mostly scientists)

~400 yrs human stem cell experience (in a field ~15 yrs old)

>600 patents (owned or licensed)

Core competencies

Creation and culture of human iPS cells


Genetic engineering of iPS cells


Differentiated cells from all three germ layers
Manufacture of human iPS cell-derived cell types


Lineage and pathway-specific markers can be introduced
Development of new differentiation protocols


Normal and disease phenotypes
Scalable production of highly purified cells
Partnership with iPS Academia Japan enables access
and support for CDI’s products in Japan
The Wall Street Journal
Gold Winner: CDI
Technology Innovation Awards 2011
5
CDI Product Portfolio

iCell® Products

iCell Cardiomyocytes – first commercial product

iCell Endothelial Cells – launched Q3 2011

iCell Neurons - launched Q4 2011

iCell Hepatocytes – “pre-commercial” release
(>13 Bn shipped since July, 2011)

Cardiomyocytes
Endothelial Cells
Neurons
Hepatocytes
MyCell Custom Products

iPS Cell Reprogramming



Episomal “footprint-free” method
Optimized for small amounts of peripheral blood
Feeder-free, defined conditions

iPS Cell Genetic Engineering

iPS Cell Differentiation


Catalog cell types (e.g. cardiomyocytes, neurons)
Custom cell types
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iCell Cardiomyocytes
• iCell Cardiomyocytes
•
•
•
•
•
•
Human iPS cell-derived
99% pure, cryopreserved, ready to use.
Available in virtually unlimited quantities
Full product solution; cells, media, protocols
Demonstrate normal human cardiac biology,
electrophysiology, and toxicity responses
Broad platform utility for discovery and preclinical
development
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CDI’s Commitment
Quality, Quantity, Purity
Key Characteristics for Adoption
Quality

Exhibit key cellular characteristics

Recapitulate normal human biology

Reproducible

Known and relevant genotype
Cell Purity
Target Cell (non proliferating)
Non-Target Cell (proliferating)
Days in Culture
Quantity

Purity
Sufficient to support HTP drug screening and safety
testing
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iCell Cardiomyocytes
Genomic and Transcript Characterization
Transcriptome Analysis
Comparative Analysis
iCell Cardiomyocytes
Gene Category
Stem cell
Novartis GNF expression
atlas
Heart expression ≥ 10X
median tissue expression
Transition
‘Primary’ Cardiomyocytes
GO analysis confirms
Cardiac-specific enrichment
Cardiomyocyte
Differentiation
Maintenance
Stable cardiac gene expression
Adapted from Babiarz et al., 2011
iCell Cardiomyocytes and adult mRNA (ambion) show
similar cardiac expression profiles
Josh Babiarz, MorganeAdapted
Ravon - Roche
from Babiarz et al., 2011
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iCell Cardiomyocytes
Protein Expression
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iCell Cardiomyocytes
Electrophysiology Characterization
Ionic Currents
INa
Ito
Spontaneous Action Potentials
ICa-L
IKr
Ma, et al, Am. J. Physiol., 2011
IK1
Gαq – α1
Isoproterenol
Phenylephrine
Gαi – m2
Carbachol
Frequency
Ifunny
Gαs – β1
Concentration (µM)
Ma, et al, Am. J. Physiol., 2011
Control
Drug
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iCell Cardiomyocytes
Functional Utility - Contractility / Ca2+ handling
Contraction
Sunny Z. Sun
Determined by edge detection
Ca2+ Transients
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Characterization Summary
 Stable genomic and
expression profile
 Expected protein localization
 Typical cardiomyocyte
electrophysiology
 Appropriate mechanical
activity
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Bioenergetics

Cellular ATP and Bioenergetic Measurements

iCell Cardiomyocyte Bioenergetic Characterization

iCell Cardiomyocyte / H9C2 Model Comparison
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Cellular ATP Production
Glycolysis
Electron Transport
Lactate by-product
Oxygen consumption
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iCell Cardiomyocytes
Mitochondrial Profile via XF Analyzer
Mitochondrial Function:
• Using the Mito Stress Kit key
parameters of mitochondrial
activity can be displayed in a
respiration profile
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Cardiac Energy Sources
(Lopaschuk et al, 2010)
Cardiomyocytes are “omnivores”:
• Can utilize fatty acids, glucose, lactate, ketones, and amino acids
• Fatty acids are the primary energy source in mature cardiomyocytes
• Mitochondrial oxidation is critical as glycolysis alone is not enough to meet
the energy needs
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iCell Cardiomyocytes
Energy Sources
iCell Cardiomyocytes can
18 survive in glucose and/or fatty acid media
Adapted from Rana et al., 2012
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iCell Cardiomyocytes
Energetics
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iCell Cardiomyocytes utilize
available energy sources
Adapted from Rana et al., 2012
19
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Model Systems
Different models, different bioenergetics
H9C2 cells – Immortalized Rat cardiac cell line
iCell Cardiomyocytes - Human iPSC-derived cardiomyocytes
Glycolysis
Fatty Acid Oxidation
iCell CMs
Oligo FCCP A/R
Rat H9C2
iCell CMs
H9C2
iCell CMs have lower
glycolytic capacity than H9C2 cells
iCell CMs have greater
FAO capacity than H9C2 cells
iCell Cardiomyocytes metabolism is more similar to native human cardiomyocytes
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than
the H9C2 model
Courtesy of R. Legmann
20
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Toxicity Screening
Media Effects on Experimental Outcome
Assess viability while inhibiting
mitochondrial function
(rotenone, antimycin, and oligomycin)
Media matters!
Context inappropriate media
can mask outcomes
Decreased viability is masked by glucose
21
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Adapted from Rana et al., 2012
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Discovery Screening
Phenotypic Screening for Cardioprotection
Cardioprotection is a key strategy for minimizing the effects of ischemia
(infarct, reperfusion, etc)
• Modulating mitochondrial function may be a key phenotypic target
• A key endpoint could be continued FAO under stress
XF Palmitate-BSA
FAO Reagent
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Discovery Screening
The Model Influences the Outcome
Determine the extent to which the model can actually utilize and modulate FAO
• Examine OCR in the presence and absence of fatty acid across models
iCell Cardiomyocytes provide an approximate 10-fold increase in FAO window
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Courtesy of R. Legmann
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Summary

iCell Cardiomyocytes recapitulate in-vivo cardiomyocyte
physiology

Can utilize FAO or glycolysis for ATP production similar to
native tissue

Provide a human-based model for mitochondrial-based
toxicity testing and phenotypic bioenergetic screening that is
more relevant than current models
24
Acknowledgements
Pfizer
Yvonne Will
Payal Rana
Sandi Engle
Sunny Sun
Seahorse Bioscience
Rachel Legmann
Brian Benoit
Cellular Dynamics
R&D
Application Support
25
Thank You
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