iCell Human Cells Exhibit Robust Metabolic Profiles

iCell Hepatocytes

iCell Cardiomyocytes

iCell human cells display a robust metabolic profile

suitable for interrogation of bioenergetic properties.

The labeled areas in the graphs above represent: 1)

basal respiration, 2) ATP production, 3) proton leak,

4) maximum respiratory capacity, 5) spare respiratory

capacity, and 6) non-mitochondrial respiration. iCell

Cardiomyocytes were plated at 20,000 cells/well for 7

days. iCell Neurons were plated at 100,000 cells/well

for 4 days. iCell Hepatocytes were plated at 25,000

cells/well for 3 days. Assay media compositions and

compound concentrations are listed in the table to the

right. Representative images of the cells in culture on

the XF Microplates are included above.

Human iPSC-Derived Cells for Modelling Cellular Bioenergetics:

Building a Metabolic Profile Using the XF Mito Stress Test

www.cellulardynamics.com Madison, WI USA (608) 310-5100

S. Fiene, S. Einhorn, R. Llanas, G. Salvagiotto, C. Carlson, and A. Thompson Cellular Dynamics International, Inc., Madison, WI USA

Altered cellular bioenergetic events due to metabolic dysfunction have been

implicated in numerous pathophysiological conditions. The metabolic profile of

a cell can differ from one cell type to another, illustrating the importance of

selecting a physiologically relevant cell model. Primary cells can offer relevant

human biology, but issues with cell quality, availability and donor-to-donor

variability continue to limit their large-scale implementation in vitro. Cellular

Dynamics International (CDI) has developed a manufacturing pipeline to

reproducibly generate commercial quantities of high quality, highly pure

terminally-differentiated human cell types derived from induced pluripotent stem

cells (iPSCs). iCell® Cardiomyocytes, iCell Neurons, and iCell Hepatocytes are

among the first cell types produced by CDI.

In this study, the XF96 Extracellular Flux Analyzer was used to determine

whether the iCell human cells represent a physiologically relevant cell model to

study cellular metabolism. iCell Cardiomyocytes, iCell Neurons, and iCell

Hepatocytes were cultured directly on XF Cell Culture Microplates and baseline

oxygen consumption rates were measured. Following culture optimization,

oxygen consumption levels were obtained that enable the interrogation of

metabolic function with the XF Cell Mito Stress Kit. All cell types responded to

chemical modulation of mitochondrial function as expected. Using the optimized

culture and chemical compound conditions, mitochondrial ATP production and

maximum respiratory capacity were measured to establish metabolic profiles.

The data were consistent across manufacturing batches, demonstrating both

assay-to-assay and lot-to-lot reproducibility. Together, iCell human cells and the

XF Extracellular Flux Analyzer offer an excellent in vitro platform for analyzing

mitochondrial function, understanding pathophysiology, and evaluating

therapeutic interventions in biologically relevant human cell types.

Abstract

The XF Extracellular Flux Analyzers combined with iCell Cardiomyocytes, iCell

Neurons, and iCell Hepatocytes, offer a powerful tool to investigate

bioenergetics in human cells. Robust procedures for studying the metabolism of

iCell human cells with this instrument platform have been generated. The data

show that iCell human cells provide a readily available, biologically relevant,

and reproducible cell model for studying cellular metabolism. Importantly, this

study creates a foundation for more advanced studies of cellular respiration or

other metabolic endpoints.

Summary

Acknowledgements

We would like to acknowledge Julie Meito, Rachel Legmann and Brian Benoit at

Seahorse Biosciences for their expertise and guidance when generating data

presented in this poster.

The Power of iPSC Technology

iPSC-Derived Cells as a Model System

The ability to reprogram adult somatic cells (e.g. skin, blood) into iPSCs has created

tremendous interest and excitement since discovered in 2007. For decades, researchers

have relied on short-lived primary cultures or animal models to dissect the mechanisms

and pathogenesis of disease. Now, differentiated cells derived from iPSCs have the

potential to provide a readily available source of well-characterized human cells for pre-

clinical drug screening, safety testing, and disease modelling. CDI has contributed to this

field by developing a manufacturing pipeline with improved iPSC technology that

enables large-scale production of highly pure (>95%), physiologically relevant human

cells for disease research and drug development. .

The discovery that human adult cells (e.g. skin, blood) can be induced to become stem

cells capable of differentiation into any cell type in the body (e.g. heart cells, neurons,

liver cells) has launched a new era of biomedicine with unprecedented opportunities to

advance the fields of drug development, disease research, and regenerative medicine.

iPSCs offer two key advantages over other in vitro model systems:

1. iPSCs can be differentiated into any of the 200+ cell types in the human body.

2. iPSCs can be derived from any individual or patient population using non-invasive

methods.

To enable the full potential of iPSC technology, CDI has developed a manufacturing

infrastructure to enable large-scale, parallel production of high quality, highly pure

iPSC-derived human cells from panels of donors that represent ethnic/genotypic

diversity and disease phenotypes.

Any cell type

(relevant biology)

Any individual

(population diversity) iPS Cell

Cardiomyocytes

Hepatocytes

Neurons

Endothelial cells

Hematopoietic cells

Other…

CDI offers custom MyCell™ services to reprogram patient samples to iPS cells and

differentiate them to various cell types. The addition of this service delivers the robust

and scalable manufacturing capabilities of CDI to the individual investigator, providing

a plentiful and consistent source of material to begin asking more advanced questions

of human biology.

iCell Human Cells Exhibit Lot-to-Lot

Reproducibility

Oxidative consumption rates (OCR)

of two distinct lots of iCell

Cardiomyocytes, iCell Neurons, and

iCell Hepatocytes were measured

using the XF Mito Stress Kit before

and after drug treatment (Oligomycin

and FCCP). Similar levels of

response to each were obtained

across lots. Culture and assay

conditions are identical to those listed

in the previous panel’s table.

iCell Human Cells Exhibit Cell Type-

Specific Bioenergetic Profiles

Cell Type Media Formulation Drug Concentration

iCell

Cardiomyocytes

XF Assay Media

+10 mM Galactose

+ 1 mM Sodium

Pyruvate

+ 4 mM L-glutamine

Oligomycin: 1μM

FCCP: 0.5μM

Rotenone: 2μM

iCell Neurons XF Assay Media

+ 25 mM Glucose

+ 0.2 mM Sodium

Pyruvate

Oligomycin: 1μM

FCCP: 1μM

Rotenone: 0.5μM

iCell

Hepatocytes

XF Assay Media

+ 10 mM Glucose

+ 0.2 mM Sodium

Pyruvate

Oligomycin: 1μM

FCCP: 0.4μM

Rotenone: 2μM

iCell Human Cells derived from the same donor exhibit differences in

metabolism. iCell Cardiomyocytes display a large dependence on oxidative

phosphorylation relative to glycolysis as shown by the large OCR/ECAR ratio.

iCell Neurons are relatively more glycolytic when compared to iCell

Cardiomyocytes or iCell Hepatocytes as evident by the lower OCR/ECAR ratio.

These data suggest that terminally differentiated cells from the same donor

iPSCs exhibit cell type-specific metabolic profiles.

Target

Identification

Target

Validation

Compound

Screening

Lead

Optimization

Preclinical

Trials

Clinical

Trials

iCell Neurons

iCell Cardiomyocytes iCell Neurons

iCell Hepatocytes