Leptin promotes endothelial cell (EC) proliferation and angiogenesis in adult blood vessels during wound healing. Leptin also regulates the function of adult cardiomyocytes. To investigate whether leptin promotes differentiation of embryonic stem cells toward ECs or cardiomyocytes, we treated mouse embryoid bodies (EBs) with leptin and evaluated expression of specific EC and cardiomyocyte markers. Expression was quantified by immunocytochemistry and qRT-PCR in leptin treated and untreated EBs at 10-day intervals. CD31 expression was quantified by FACS. EBs were harvested and ECs were isolated by magnetic sorting. Cells were then labeled with green fluorescent protein under the control of ubiquitin C promoter and implanted in quail chorioallantoic membranes (CAMs) to evaluate vessel formation. More robust vascular networks and beating clusters were found in leptin-treated EBs. FACS quantification demonstrated higher expression of CD31 in leptin-treated EBs vs. controls (65 vs. 17%).

Significant up-regulation of ECs and cardiomyocyte specific genes were found in leptin-treated EBs.

Interestingly, some CD31 positive cells also expressed cardiotin indicating that leptin may promote proliferation and survival of cardiovascular progenitors. Isolated cells were cultured and implanted on CAMs. Vascular integration of labeled cells derived from leptin-treated EBs was observed in nascent CAM blood vessels. However, beating structures in this in vivo model did not persist. In summary, leptin promotes the differentiation of ECs and cardiomyocytes by up-regulating specific genes and enhancing the formation of a robust microvasculature as well as beating clusters in mouse EBs.

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