VEGF promotes endothelial cells differentiation from human embryonic stem cells mainly through PKC-ε/η pathway.

Human embryonic stem cells (hESCs) have unlimited proliferation capacity and can differentiate into most types of somatic cells. We previously described that the overexpression of FLI1 as well as the activation of protein kinase C (FLI1-PKC) could rapidly and efficiently differentiate hESCs into endothelial cells (ECs). However, the relationship between vascular endothelial growth factor (VEGF) and PKC in hESC-EC differentiation is debated, and the roles of different PKC isoforms in hESC-EC differentiation remain unknown. In this study, after 2 days of induction, we found that the overexpression of FLI1 and the addition of VEGF-A to hESCs (FLI1-VEGF) could generate 19.6% +/- 5.4% ECs. The induction efficiency of ECs was reduced from 72.3% +/- 7.6% to 37.9% +/- 4.9% following the addition VEGF-A siRNA to the FLI1-PKC system. However, the induction of ECs was nearly completely abrogated following the addition of the pan-PKC inhibitor to the FLI1-VEGF, FLI1-PKC, or other systems. The above results suggested that VEGF can partially replace PKC, but PKC plays a more critical role downstream of VEGF during hESC-EC induction. To further investigate which PKC isoform was mainly involved in converting hESCs to ECs, siRNAs were used to knock down nine PKC isoforms in the FLI1-PKC system. Only the knockdown of PKC-e or PKC-eta significantly decreased the induction efficiency of ECs to 51.1% +/- 5.8% or 50.3% +/- 5.1%, respectively. PKC-e/eta siRNA could suppress EC induction in other differentiation systems. Moreover, RNA-seq and quantitative polymerase chain reaction analysis also showed that only the expression of PKC-e and PKC-eta was robustly upregulated during hESC-EC induction. In summary, our results suggested that VEGF promoted the differentiation of ECs derived from hESCs, which mainly depended on PKC, specifically the PKC-e and PKC-eta pathways.