Engineering cardiomyocytes from diverse human pluripotent stem cell lines for cardiac cell therapy with optimized cell yield and efficiency
Parikh, Abhirath S.
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Human embryonic and induced pluripotent stem cells (collectively 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 specific cardiac muscle cell-types across different hPSC lines having varying lineage-specific differentiation propensities. This requires a detailed understanding of signaling pathways and differentiation mechanisms in context of several ambient conditions such as cell epigenetic profile, endogenous signaling, and cell differentiation state. Therapeutic realization also hinges on the transplantation of sufficient numbers of cells (approximately 1.5x10 9 per damaged myocardium ) pointing to the need for pertinent large-scale bioprocesses. In this study a novel, hormone-free, xeno-free medium is developed that is devoid of signaling activity. Since contemporary xeno-free formulations contain agents with signaling activity (such as growth hormone, retinoic acid, corticosterone, LiCl, ascorbic acid) that are reported to interfere with cardiac differentiation, proper assessment of signaling mechanisms is not feasible. Therefore a factorial design consisting of 73 conditions is employed a develop a medium comprising of specific combination of basal medium, iron-carrier (holo-transferrin), reducing agent (selenium), and a novel cocktail to increase cell growth and viability (termed XFM cocktail) containing optimized levels of lipids, aminoacids and vitamins. The medium developed here is utilized to investigate signaling mechanisms governing cardiomyocyte differentiation of hPSCs. The simple formulation of the medium offers significant economic advantages over other xeno-free media for a large scale differentiation process. Finally, since the medium is free from xenogeneic and allogeneic agents (such as albumin), clinical translation is feasible preventing the risk of transferring animal-derived pathogens and non-native proteins that can trigger adverse foreign-body immune reactions and possible implant rejection. This is the first report of accurate assessment of majority of physiologically-relevant signaling pathways elucidating their individual roles in cardiac differentiation as well as formation of closely-related lineages. By employing a design-of-experiment approach, an efficient path of hPSC-to-cardiomyocyte differentiation is discovered comprising of two steps. A synergistic combination of BMP and Wnt3A treatment with endogenous FGF and Nodal signaling is determined for obtaining an intermediate progenitor-like stage termed mesoderm-oriented primitive streak (MePS) that significantly increases differentiation efficiency. Adjustment of treatment concentration and duration during the first step and appropriate time of starting the second step allows for efficient conversion of MePS cells to cardiomyoctes in a total of 6 days of differentiation. This optimization renders the method highly reproducible that translates across the most diverse set of hPSC lines with varied lineage-specific differentiation propensities. The efficiency of hPSC-to-cardiomyocyte conversion is >90% and the cell yield is the highest reported yet in the literature providing 23 cardiomyocytes per stem cell seeded or 0.9 million cardiomyocytes/cm 2 . First beating is seen as early as day 6 of differentiation and by day 8 there is interconnected and synchronized beating activity with majority of cells beating in each dish. By day 16 of differentiation, formation of bi-nucleated cells is observed indicating maturation toward adult-like cardiomyocytes. Distribution of cardiac cell-types is determined by electrophysiological measurements to be ∼60% ventricular and ∼40% atrial and sinoatrial node. ^ Investigation of molecular mechanisms in the first stage reveals a crosstalk between BMP and Wnt pathways providing novel scientific information regarding mechanisms governing efficient cardiomyocyte differentiation. In the next stage, pathways are elucidated that selectively pattern primitive streak cells to different mesoderm derivatives such as smooth muscle, hematopoietic, blood and cardiac. Initial results demonstrate increased cardiac gene expression and modulation of atrial and ventricular markers providing avenues for fine-tuning mesoderm into different cardiac cell-types and possibly for increasing yield of differentiation. The outcome of this thesis is expected to facilitate the development of stem cell-based therapies for the heart.