VEGF promotes endothelial cells differentiation from human embryonic stem cells mainly through PKC-ε/η pathway.
VEGF promotes endothelial cells differentiation from human embryonic stem cells mainly through PKC-ε/η pathway.
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VEGF主要通过PKC-δ/δ途径促进人胚胎干细胞向内皮细胞分化。
DOI：
10.1089/scd.2019.0172
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发表时间：
2019-11
影响因子：
4
通讯作者：
Liang Hu
中科院分区：
文献类型：
--
作者：
Hao Zhao;Mengge Li;Qi Ouyang;Ge Lin;Liang Hu
人胚胎干细胞（hESCs）具有无限的增殖能力，可以分化为大多数类型的体细胞。我们之前报道过表达FLI1和激活PKC （FLI1-PKC）可以快速有效地将hESCs分化为内皮细胞（ECs）。然而，VEGF和PKC在hESC-EC分化中的关系尚存在争议，PKC不同亚型在hESC-EC分化中的作用尚不清楚。在本研究中，我们在诱导2天后发现，过表达FLI1并在hESCs中添加VEGF-A （FLI1- vegf）可产生19.6±5.4%的ECs。在FLI1-PKC系统中加入VEGF-A siRNA后，ECs的诱导效率从72.3±7.6%降低到37.9±4.9%。然而，在FLI1-VEGF、FLI1-PKC或其他系统中加入泛pkc抑制剂后，ECs的诱导几乎完全被取消。上述结果提示，在hESC-EC诱导过程中，VEGF可以部分替代PKC，但PKC在VEGF下游发挥更关键的作用。为了进一步研究哪一种PKC异构体主要参与hESCs向ec的转化，研究人员使用sirna敲除FLI1-PKC系统中的9种PKC异构体。PKC-ε和PKC-η的敲除显著降低了ECs的诱导效率，分别为51.1±5.8%和50.3±5.1%。PKC-ε/η siRNA在其他分化系统中也能抑制EC的诱导。此外，RNA-seq和qPCR分析也显示，在hESC-EC诱导过程中，只有PKC-ε和PKC-η的表达明显上调。综上所述，我们的研究结果表明，VEGF促进hESCs衍生的ECs的分化，主要依赖于PKC，特别是PKC-ε和PKC-η途径。
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 PKC (FLI1-PKC) could rapidly and efficiently differentiate hESCs into endothelial cells (ECs). However, the relationship between 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 knockdown 9 PKC isoforms in the FLI1-PKC system. Only the knockdown of PKC-ε or PKC-η significantly decreased the induction efficiency of ECs to 51.1±5.8% or 50.3±5.1%, respectively. PKC-ε/η siRNA could suppress EC induction in other differentiation systems. Moreover, RNA-seq and qPCR analysis also showed that only the expression of PKC-ε and PKC-η 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-ε and PKC-η pathways.
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