Abstract
Although the study on the regulatory mechanism of endothelial differentiation from the perspective of development provides references for endothelial cell (EC) derivation from pluripotent stem cells, incomplete reprogramming and donor-specific epigenetic memory are still thought to be the obstacles of iPSCs for clinical application. Thus, it is necessary to establish a stable iPSC-EC induction system and investigate the regulatory mechanism of endothelial differentiation. Based on a single-layer culture system, we successfully obtained ECs from porcine iPSCs (piPSCs). In vitro, the derived piPSC-ECs formed microvessel-like structures along 3D gelatin scaffolds. Under pathological conditions, the piPSC-ECs functioned on hindlimb ischemia repair by promoting blood vessel formation. To elucidate the molecular events essential for endothelial differentiation in our model, genome-wide transcriptional profile analysis was conducted, and we found that during piPSC-EC derivation, the synthesis and secretion level of TGF-β as well as the phosphorylation level of Smad2/3 changed dynamically. TGF-β-Smad2/3 signaling activation promoted mesoderm formation and prevented endothelial differentiation. Understanding the regulatory mechanism of iPSC-EC derivation not only paves the way for further optimization, but also provides reference for establishing a cardiovascular drug screening platform and revealing the molecular mechanism of endothelial dysfunction.