Increased Cardiomyocyte Differentiation from Human Embryonic Stem Cells in Serum‐Free Cultures

Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is low. We routinely induce cardiomyocyte differentiation of the HES‐2 cell line by coculture with a visceral endoderm‐like cell line, END‐2, in the presence of 20% fetal calf serum (FCS). In this study, we demonstrate a striking inverse relationship between cardiomyocyte differentiation and the concentration of FCS during HES‐2‐END‐2 coculture. The number of beating areas in the cocultures was increased 24‐fold in the absence of FCS compared with the presence of 20% FCS. An additional 40% increase in the number of beating areas was observed when ascorbic acid was added to serum‐free cocultures. The increase in serum‐free cocultures was accompanied by increased mRNA and protein expression of cardiac markers and of Isl1, a marker of cardiac progenitor cells. The number of beating areas increased up to 12 days after initiation of coculture of HES‐2 with END‐2 cells. However, the number of α‐actinin–positive cardiomyocytes per beating area did not differ significantly between serum‐free cocultures (503 ± 179; mean ± standard error of the mean) and 20% FCS cocultures (312 ± 227). The stimulating effect of serum‐free coculture on cardiomyocyte differentiation was observed not only in HES‐2 but also in the HES‐3 and HES‐4 cell lines. To produce sufficient cardiomyocytes for cell replacement therapy in the future, upscaling cardiomyocyte formation from hESCs is essential. The present data provide a step in this direction and represent an improved in vitro model, without interfering factors in serum, for testing other factors that might promote cardiomyocyte differentiation.