Abstract
Objective Preliminarily study the mechanism of the proliferation of mouse Induced pluripotent stem cells (miPSC)-endothelial cells (EC) by lentivirus-mediated yes-associated protein (YAP) gene overexpression. Methods Induce miPSC to EC. The pPGK-2 Flag YAP plasmid was packaged into mature lentivirus to infect 293FT cells. The supernatant of the infected cells was harvested to infect miPSC-EC and a stably infected cell lines were screened by puromycin, in which YAP expression was detected using Western blotting. Detect and compare the protein expression level of Phosphatase and tensin homolog deleted on chromosome ten (PTEN) and increased the phosphorylation of protein kinase B (Akt), phosphorylated-protein kinase B (p-Akt) between miPSC-EC and miPSC-EC/YAP. Cell proliferation ability was determined by methyl thiazolyl tetrazolium assay (MTT). Results The cells induced from miPSC have the same morphology of endothelial cells. Almost all cells expressed endothelial cell marker CD31 (Platelet endothelial cell adhesion molecule-1). The YAP protein in miPSC-EC/YAP was obviously expressed, and while the YAP protein in nontransfected cells was almost not expressed. Overexpressing YAP in miPSC-EC resulted in the decreased expressing of PTEN (0.635±0.017 vs. 0.879±0.034, P=0.023) and the increased expressing of P-Akt (0.450±0.025 vs. 0.073±0.018, P=0.007). The optical density (A) value of YAP group was higher than that in control group at time points of 24 h, 48 h and 72 h after synchronization (0.113±0.004 vs. 0.077±0.004, P=0.000; 0.368±0.010 vs. 0.199±0.006, P=0.000; 0.716±0.004 vs. 0.418±0.018, P=0.000). Conclusion EC derived from miPSC was successfully induced. The cell line miPSC-EC/YAP with stable YAP gene overexpression was established successfully. Overexpressing YAP inhibited the expression of PTEN. It indicated that overexpressing YAP may promote the proliferation of miPSC-EC through the phoshoinositide-3-kinase/protein kinase B (PI3K-Akt) signal pathway. Key words: Yes-associated protein; Induced pluripotent stem cells; Endothelial cells; Phoshoinositide-3-kinase/Protein kinase B
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