(145d) Directed Differentiation of Embryonic Stem Cells to Cardiomyocytes in a Bioreactor

Heart diseases are top ranked as causes of morbidity and mortality in the US and most developed countries. Myocardial infarction is associated with significant cardiomyocyte death and permanently impaired cardiac function. Widespread utility of heart transplantation is hindered due to severe shortage of donor organs. Hence, the search for alternative sources of heart cells including embryonic stem cells (ESCs) has intensified in recent years.

In this study we explored the differentiation of ESCs towards cardiomyocytes in the absence of serum and with factors involved in embryonic heart development. Current methods for ESC-to-heart muscle cell differentiation rely on the use of serum which makes challenging the control of ESC specification, for example, via the addition of physiologically relevant agents. The resulting cell populations are heterogeneous and contain only minute fractions of cells displaying cardiomyocyte markers.

We have identified conditions using defined serum-free medium and TGF-β ligands (mainly bone morphogenetic proteins; BMP) for directing the differentiation of mouse ESCs (mESCs) to cardiomyocyte-like cells. When cultured in dishes, mESCs formed beating foci whereas cells not treated with BMP failed to organize into contractile areas. The fraction of foci beating reached 90% within 2 weeks and remained constant thereafter. The cells displayed cardiac α-actinin, cardiac troponins and the cardiac transcription factor Nkx2.5 as assessed by reverse transcription-PCR (RT-PCR) and the immunostaining.

Given the need for generating heart cells in adequate quantities for clinical uses a method was subsequently developed for directing the differentiation of mESCs in a stirred-suspension bioreactor without serum. The expression of early mesoderm and cardiac genes was assessed by quantitative PCR (qPCR). Brachyury was up-regulated by day 7 but its expression declined as the differentiation progressed towards more mature cardiac cells. Concomitantly, the expression of GATA4 increased reaching a 5.4±0.4 fold higher level compared to control cells. Alpha-myosin heavy chain (α-MHC) and atrial natriuretic factor (ANF) were strongly expressed in differentiating cells but were absent in the undifferentiated ESCs. Cells treated with BMP were also immunoreactive for cardiac troponin I (cTnI), cardiac transcription factor Nkx2.5, and α-actinin. In addition, ESC-derived heart muscle cells responded to treatments with pharmacological stimuli known to modulate the contractility of normal cardiomyocytes. For example, treatment with a phosphodiesterase inhibitor increased the beating rate of the contractile regions in a dose-dependent manner suggesting that a cAMP-dependent mechanism mediates the observed beating activity similar to native cardiomyocytes. The use of a suspension bioreactor for the differentiation of ESCs resulted in the generation of higher numbers of cardiac-like cells per unit of medium volume. Therefore, such bioreactor systems may be used for the production of heart cells from ESCs in a more efficient and economic fashion.

Current efforts are focused on translating these findings to human ESC (hESC) differentiation aiming at the development of a scalable system for the generation of cardiomyocytes for cell therapies. Differentiation of hESCs has been carried out in static cultures resulting in beating regions. Compared to undifferentiated ESCs, cells within these regions express heart cell-specific genes, such as ANF, human α-MHC and β-MHC as tested by qPCR. Heart proteins such as α-actinin, Nkx2.5 and cTnI were also identified by immunostaining. Lastly, differentiated hESCs responded to pharmacological stimuli by altering their beating pattern.

Development of methods for directed differentiation of hESCs in bioreactors as described here for mESCs, will have a significant impact in enabling hESC-based technologies and therapies in the clinical setting.