Differentiation Of Embryonic Stem Cells Into Endothelial (Progenitor) Cells And Their Characterization In Comparison With The Endothelial Cells Derived From Tissues

Vascular endothelial cell damage is related to the occurrence and development of many cardiovascular and cerebrovascular diseases,and has become an important research object in the field of life sciences and pharmacy.Cardiovascular disease(CVD)is usually caused by endothelial dysfunction and is one of the leading causes of death in the world.The lack of a sufficient number of functioning endothelial cells(ECs)is one of the reasons preventing the treatment of cardiovascular disease by cell replacement.Human pluripotent stem cells include human embryonic stem cells(h ESCs)and human induced pluripotent stem cells(hi PSCs),which can provide the opportunity to produce a large number of functional endothelial cells in vitro,which can be used for transplantation,drug screening or research.However,currently h ESCs-derived ECs have a variety of differentiation schemes,and the research on the characteristics of endothelial cell types after differentiation has not been completed.In particular,the effects of different differentiation methods and basic culture media on the differentiation of embryonic stem cells into endothelial cells and the differences in cell characteristics after differentiation have not been fully studied.1.Isolation,culture and identification of primary endothelial cells.The present study has used a simple and rapid isolation and culture system for human umbilical cord artery endothelial cells(HUAEC)and umbilical cord vein endothelial cells(HUVEC)with high purity.Multiple facets and dynamic changes in biological characteristics including morphology,proliferation,tube formation,surface antigen and the expression of specific genes were analyzed for both HUAEC and HUVEC under the same culture conditions in vitro.We found that there was no significant difference in the morphology,tube forming ability,surface antigen(CD144,CD31,CD309,CD133,CD34)between HUAEC and HUVEC for a long-term continuous subculture,although HUAEC showed a higher proliferation activity than HUVEC.For freshly isolated HUAEC and HUVEC,the expression levels of the specific genes for artery and vein respectively were significantly different(HUAEC high expression in EFNB2,DLL4,NRP1,CXCR4;HUVEC high expression in EPHB4,COUP-TFII).However,during the prolonged culture period(after Passage 6)HUAEC lost then its specific gene expression,whereas HUVEC remained its high expression of the specific genes.In conclusion,HUVECspecific expression genes EPHB4 and COUP-TFII can be used as reliable identification markers for distinguishing human umbilical cord artery-or umbilical cord vein-derived endothelial cells cultured in vitro.2.Characterization of macrophage-like cells differentiated from THP-1 by CSFs.3.Differentiation of human embryonic stem cells into endothelial cells.In this study,human embryonic cells were differentiated into endothelial cells by embryoid body differentiation.The differentiated h ESCs-ECs expressed endothelial cell surface antigens CD31 and CD144,and formed tubular structures on matrigel in vitro.Stemline ? has higher differentiation efficiency than the Stempro 34 medium as previously reported.Compared with Stemline ? and Stempro 34,ECM and EGM-2 showed better cell viability and higher CD31 expression,but there was no obvious clustering in flow cytometry.3.Differentiation of human embryonic stem cells into endothelial progenitor cells.By comparing embryoid body differentiation with monolayer adherent differentiation,it was found that h ESCs-EPCs with monolayer adherent differentiation are more oval in shape and more uniform in shape.Adherently differentiated h ESCs-EPCs have significantly lower tube-forming capacity than h ESCs-ECs with embryoid body differentiation.The obtained endothelial cells showed a markedly high expression of the endothelial progenitor cell marker CD133,and they had a tendency to differentiate into mature endothelial cells after passage.In addition,h ESCs-EPCs derived from adherent differentiation showed a higher number of passages of proliferative viability.Monolayer adherence differentiation is consistent with embryoid body differentiation.Endothelial cells derived from all differentiation schemes have not yet been completely destined for arterial endothelial or venous endothelial cells,and are in a more primitive state of endothelial cells or endothelial progenitor cells.But it can be characterized as type II endothelial cells,which has the property of inhibiting smooth muscle cell proliferation.