Abstract 3873: Endothelial Cells Promote Embryonic Stem Cell-Derived Cardiomyocytes Maturation in vitro and in vivo
Background Embryonic stem cells (ESCs) can be induced to differentiating into a cardiomyocyte lineage and thus may provide a stem cell source for myocardial repair. However, ESCs-derived cardiomyocyte-committed cells are difficult to isolate, are highly heterogeneous in cell morphology and do not contract synchronically. In other words, they are immature cardiomyocyte precursors (CPs). In the present study, we sought to develop a protocol for the purification of ESC-derived CPs (ESC-CPs) and to investigate if endothelial cells, which mediate the late stage of embryonic cardiogenesis, may promote the function and maturation of ESC-CPs.
Methods and Results We used a mouse ESC line with a cardio-specific α-myosin heavy chain-driven enhanced green fluorescence protein (eGFP) reporter to allow visualization and isolation of ESC-CPs following hanging drop method for 6 days. With vitamin C supplement in the hanging drops, there were ~5% of ESCs differentiating into CPs, with no effect of the yield of ESC-CPs by the coculture with endothelial cells or fibroblasts. ESC-CPs survived the cell sorting procedure when collected in a serum-enriched medium with an initial survival rate ~ 98% (the immediate post-sorting apoptosis rate was 2.24±0.31%). Cocultured with endothelial cells or fibroblasts did not change the survival rate of ESC-CPs after 24 hours of sorting, however, cocultured with endothelial cells, but not with fibroblasts, significantly increased the maturation indexes of CPs, including the cell size, axis alignment, sarcomere organization and length, contraction rate, and the expression of gap junction protein connexin-43. The increase of ESC-CPs by endothelial cells required direct cell-to-cell contact, as demonstrated by a transwell coculture assay. Finally, intramyocardial injection of ESC-CPs with endothelial cells, but not with fibroblasts, in immuno-compromised mice. improved the survival, alignment and connexin-43 expression of ESC-CPs after 7 days.
Conclusion Endothelial cells promote the maturation of ESC-CPs in vitro and in vivo. This strategy may aid in the application of using ESC-CPs for cardiac regeneration.
This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).