(453c) Engineering Cardiomyocytes From Diverse Human Pluripotent Stem Cell Lines For Cardiac Cell Therapy With Optimized Cell Yield and Efficiency

Human embryonic and induced pluripotent stem cells (known as pluripotent stem cells or hPSCs) can be a renewable source of cardiomyocytes for treating heart diseases which are leading causes of morbidity and mortality. Recent advances in the generation of patient-specific induced PSCs greatly improve the chances of clinical success. Yet, robust methods are still under development for producing cardiomyocytes across different hPSC lines having varying lineage-specific differentiation propensities. Therapeutic realization also hinges on the transplantation of sufficient numbers of cells (approximately 1.5x10⁹ per damaged myocardium (1)) pointing to the need for pertinent large-scale bioprocesses. To resolve these issues, we have assessed particular signaling pathways related to heart tissue formation such as those induced by bone morphogenetic proteins (BMPs), Wnt, Nodal/Activin, and FGF ligands for efficient cardiac differentiation, and investigated methods for improving the yield of cardiomyocytes across a variety of hPSC lines.

A novel serum-free, defined medium formulation was developed by accounting for the signaling mechanisms linked to cardiogenesis aiming at increasing the reproducibility of in vitro commitment of hPSCs to heart cells. Human PSCs were efficiently directed in 48 hours to mesoderm-oriented primitive streak using BMP and Wnt ligands. Treated cells were characterized by high expression levels of Brachyury (T), MIXL1, EOMES and MESP1 assessed by qPCR and immunofluorescence. The total fraction of T⁺ cells determined by flow cytometry was more than 95%. Subsequently, manipulation of physiologically-relevant signaling pathways that interact and participate in mesoderm patterning such as Wnt, Nodal/activin, BMP and FGF resulted in different mesoderm derivatives. Therefore, a specific combination of these pathways was utilized to coax these cells further along cardiovascular lineages expressing KDR, cKIT, Nkx2.5, GATA4 and MEF2C. Beating cardiomyocytes emerged within a week and the appearance of beating clusters increased till 12 days of differentiation displaying several beating foci (>10 per cm²) that corresponded to beating of more than 80% of the cells in each dish. During this period, cardiac markers such as β-MHC, TBX20, MLC2a and ANF were strongly upregulated. Expression was higher than that seen in cells treated with conventional protocols for cardiogenic differentiation. Upon further maturation, corresponding upregulation was observed by western blot analysis and immunofluorescence of cardiac proteins such as NKX2.5, GATA4, MEF2C, α-actinin, cardiac troponins and the cardiac gap junction protein connexin-43. When subjected to electrophysiological measurements, cells displayed cardiac action potentials indicating approximately 60% ventricular cells and a mix of atrial and sinoatrial node (pacemaker) cells. Connections among neighboring contractile foci in each culture were observed leading to integrated beating activity. Single cells were also capable of displaying contractions and their coupling with neighboring cells led the emergence of binucleated cell clusters typical of cardiomyocytes. Their contractile activity was modulated in a dose-dependent, organotypic fashion by incubation with diltiazem, isoproterenol and phosphodiesterase inhibitors. Up to 90% of committed cells expressed particular cardiac markers as assessed by flow cytometry yielding a significant number of cardiac cells per stem cell initially seeded.

An optimized protocol for cardiogenic differentiation of hPSCs was developed based on the rational manipulation of the core signaling pathways involved in cardiac cell commitment. Application of the method improved the efficiency and yield across different hPSC lines. Ongoing work concentrates on translating our findings to the generation of functional cardiomyocytes from hPSCs in scalable cultivation systems (2). The outcome of our studies is expected to facilitate the development of stem cell-based therapies for heart diseases.

Acknowledgements: Funding support has been provided by the National Institutes of Health (NHLBI, R01HL103709) and the New York Stem Cell Science Trust (NYSTEM, contract C024355) to EST. AP is a recipient of the Mark Diamond Research Fund award.

References:

1.           Jing D, Parikh A, Canty JM, Tzanakakis ES. Stem Cells for Heart Cell Therapies. Tissue Eng Part B Rev.14:393-406. 2008.

2.           Jing D, Parikh A, Tzanakakis ES. Cardiac cell generation from encapsulated embryonic stem cells in static and scalable culture systems. Cell Transplant.19:1397-412. 2010.