Embryonic epicardium is crucial for normal heart morphogenesis. The tissue originates from the proepicardial organ, with cells migrating to form the outer mesothelial layer, giving rise to non-myocyte cells during early heart development. The epicardium's formation is crucial for myocardial growth and compaction, as well as the embryonic heart's coronary vessel development. These capabilities are useful for engineering mature myocardial tissue in vitro. Recent research shows that human-induced pluripotent stem cells can differentiate into embryonic epicardial cells through specific signaling pathways during in vitro development. Here, we present a simple differentiation protocol to derive a premature form of epicardial cells (or pre-epicardial cells, PECs) from the lateral plate mesoderm, followed by stimulation with BMP4, VEGF, and retinoic acid. The differentiated PECs express WT-1 and ZO-1, exhibiting typical epicardial cell features, including the ability to undergo epithelial-mesenchymal transition and differentiate into fibroblast and smooth muscle cells. Furthermore, PECs can aggregate and stimulate cardiomyocyte proliferation in coculture via the insulin-like growth factor-2 signaling pathway. Furthermore, PECs also show the ability to aggregate and induce proliferation of cardiomyocytes in coculture through the insulin-like growth factor-2 signaling pathway. In a three-dimensional culture, PECs promote the formation of vessel-like structures in CM microtissues, complete with defined smooth muscle and endothelial linings. These characteristics suggest the significant role of PECs in organizing cardiac tissue, presenting promising applications in tissue engineering and therapeutics.

 

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