Industrialized Production of Human iPSC-Derived Cardiomyocytes for Use in Drug Discovery and Toxicity Testing - Pharmaceutical Technology

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Industrialized Production of Human iPSC-Derived Cardiomyocytes for Use in Drug Discovery and Toxicity Testing
The authors describe an industrialized process for the manufacture of iPSC-derived human cardiomyocytes. This article is part of a special issue on Bioprocessing and Sterile Manufacturing.


Pharmaceutical Technology
Volume 34, pp. s8-s15

Cardiomyocyte differentiation and purification


Figure 2
Another barrier to the use of iPSC-derived cell types for drug discovery is the production of highly purified terminally differentiated cell types. Random in vitro differentiation, for example through the embryoid body method, is inherently inefficient. Our proprietary directed differentiation method has been able to increase the efficiency of cardiomyocyte differentiation by orders of magnitude (see Figure 2). However, the final cell product requires greater cell purity in order to ensure that an observed experimental response is due to an effect on the target cell type and not other contaminating cells.

CDI has achieved greater cell purity with the following procedure. Prior to iPSC clonal expansion, genes encoding antibiotic resistance and red fluorescent protein (RFP) under control of a pan-cardiac promoter are introduced into the iPSCs through homologous recombination. The use of homologous recombination can target a location on the host chromosome and insert an element of choice, ensuring minimal disruption of endogenous genes. After curation and quality control, the iPSC clone carrying the selectable marker is expanded in multiple CellSTACKs to produce sufficient iPSCs for the cardiomyocyte differentiation. Once expanded, iPSCs are harvested and seeded into spinner flasks with the presence of small molecules and growth factors. Beating aggregates of cardiomyocytes are observed within days after withdrawal of the growth factor cocktail.

Through this method alone, we have been able to routinely achieve cardiomyocyte purities greater than 50% (see Figure 2). As the cardiomyocytes contain the exogenous antibiotic resistance gene while "contaminating" non-cardiomyocytes do not, the population purity is subsequently increased to approximately 100% through exposure to antibiotic. RFP expression confirms the increase in purity. Purified cardiomyocytes are matured in cardiomyocyte maintenance medium prior to cryopreservation. Following re-animation from the cryopreserved state, the high level of cardiomyocyte purity is maintained by culturing the cells in a proprietary medium developed by CDI that eliminates the growth of proliferating (i.e. non-cardiomyocyte) cells and allows the end-user to maintain pure cardiomyocyte cultures in vitro for weeks.

Human iPSC-derived cardiomyocyte characterization

Although purified iPSC-derived cardiomyocytes have the physical appearance of cardiomyocytes and aggregations of the cells exhibit synchronous contractile activity (i.e., the cells beat), we tested the cells' biochemical and electrophysiological properties to determine their utility for drug development and toxicity testing.


Figure 3
Cardiomyocytes manufactured using industrial-scale culture and our cell differentiation process expressed several genes found specifically in cardiomyocytes (see Figure 3). Gene expression of human cardiomyocyte mRNAs was tested using real-time PCR between 0 and 32 days post initiation of differentiation. Gene expression was quantified using Taqman (Applied Biosystems, Carlsbad, CA) gene expression assays for a number of transcription factors, cytoskeletal components, and ion channels. Levels of the stem cell transcription factor Oct-4 decreased during differentiation of iPSC-derived cardiomyocytes, while all cardiomyocyte-specific mRNAs expression levels increased.


Figure 4
The presence of cardiac-specific protein markers was also investigated. iPSC-derived cardiomyocytes were shown to express the cardiomyocyte-specific proteins sarcomeric alpha actinin and troponin I (see Figure 4).


Figure 5
Cardiomyocyte subtypes of the heart have distinctive electrophysiological profiles that can be characterized by, among other items, early depolarization events (Phase 4 depolarization) and the action potential duration. As shown in Figure 5, action potentials produced by individual iPSC-derived cardiomyocytes recapitulate qualities of action potentials of native nodal, atrial, and ventricular cardiomyocytes.

Human iPSC-derived cardiomyocyte pharmacology


Figure 6
iPSC-derived cardiomyocytes should also mimic responses to chemical perturbations seen in normal human cardiomyocytes. Electrophysiological responses of cardiomyocytes were tested against exposure to E-4031, a blocker of the human Ether-�o-go Related Gene (hERG) potassium channels that are primarily expressed in the heart, and to nifedipine, a blocker of the voltage-dependent L-type calcium channel also expressed in the heart. In both cases, the change in shape of the cardiac action potential from single cells recorded with the patch clamp or the shape of the field potential of a population of beating cardiomyocytes recorded with a microelectrode array exhibit the expected prolongation or shortening of the signal as expected based on work with native cells and tissue (see Figure 6).


Figure 7
iPSC-derived cardiomyocytes showed sensitivity to known cardiotoxic compounds (see Figure 7) affecting biochemical processes. Cardiomyocytes exposed to staurosporine for 16 hours showed half maximum effective values (EC50 values) of 575 nM for the live assay and 482 nM for the dead assay. Caspase activity was measured after six hours of drug exposure and demonstrated an EC50 of 585 nM, once again demonstrating the expected responses of native cardiomyocytes.

Conclusion

Cellular Dynamics (Madison, WI) has developed a highly standardized, scalable process to manufacture human iPSC-derived cardiomyocytes. Using this process, the company launched its iCell Cardiomyocytes product line in December 2009, whereby we can produce the hundreds of billions of human cardiomyocytes needed by the pharmaceutical industry for drug-toxicity testing.

These iCell Cardiomyocytes demonstrate molecular and physiological properties and show sensitivity to cardiotoxic compounds expected of human cardiomyocytes. The ability to consistently produce large numbers of high quality and high purity cardiomyocytes offers the pharmaceutical industry a new tool to better predict the cardiotoxicity of new drug candidates. It is expected that these cells will allow pharmaceutical companies to more efficiently select drug candidates while reducing the likelihood that cardiotoxic activity of these compounds will manifest themselves late in development or after regulatory approval and market launch.

Blake Anson* is iCell Cardiomyocyte product manager, Emile Nuwaysir is COO, Brad Swanson is director of cell biology product development, and Wen Bo Wang is director of process sciences, all at Cellular Dynamics International, 525 Science Drive, Madison, WI 53711.

*To whom all correspondence should be addressed.

References

1. J. A. Thomson et al., Science 282 (539), 1061–1062 (1998).

2. K. Takahashi and S. Yamankai, Cell 126, 663–676 (2006).

3. J. Yu et al., Science 318 (5858): 1917–1920 (2007).

4. K. Takahashi et al., Cell 131, 861–872 (2007).

5. J. Q. He et al., Circ. Res. 93, 32–39 (2003).

6. J. Zhang et al., Circ. Res. 104 (4) e30–41 (2009).

7. G. Narazaki et al., Circulation 118 (5),498–506 (2008).

8. N. Maherali and K. Hochedlinger, Cell Stem Cell 3, 595–605 (2008)

9. J. Yu et al., Science 324 (5928): 797–801 (2009).

10. B. Feng et al., Cell Stem Cell, 4, 301–312 (2009).


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