Establishment Of Differentiation Method Of Arterial And Venous Endothelial Cells Using Human Pluripotent Stem Cell Fluorescence Reporter System

Human pluripotent stem cells(hPSCs)have the potential to differentiate into endothelial cells(ECs),which represent a sufficient cell source for the cell therapy of cardiovascular diseases,in vitro disease modeling and construction of tissue engineering blood vessels.However,hPSC-derived ECs that obtained by current methods showed obvious heterogeneity and immaturity.The characteristics of arterial endothelial cells(AECs)and venous endothelial cells(VECs)can’t be clearly distinguished,and it is difficult to satisfy the requirements for specific AECs or VECs in practical applications.Therefore,it is urgent for scientists to establish efficient methods for specific VEC and AEC differentiation from hPSCs.EphrinB2 protein(EFNB2)and Ephrin TypeB receptor4(EPHB4)have been recognized as the molecular markers of AECs and VECs,respectively.In order to effectively identify AEC and VEC subpopulations,we generated two hPSC lines which carried a fluorescent reporter system for AECs(H1-EFNB2mCherry/+hPSC line)or VECs(H1-EPHB4eGFP/+hPSC line),respectively,by CRISPR/Cas9 technology.Both H1-EFNB2mCherry/+hPSCs and H1-EPHB4eGFP/+hPSCs showed high expression of the pluripotency markers including SOX2,NANOG and OCT4,and can differentiate into cells derived from all three germ layers.Karyotype analysis showed no abnormal chromosomal structure in these cells.During the process of EC differentiation,some cells derived from H1-EFNB2mCherry/+hPSCs and H1-EPHB4eGFP/+hPSCs displayed green or red fluorescence signals under the fluorescence microscope,and the mRNA levels of mCherry and eGFP were also positively correlated with their markers,respectively.Therefore,the fluorescence reporter system established in this study represents a favorable tool for the development of specific differentiation methods for AECs and VECs.The H1-EFNB2mCherry/+hPSCs and H1-EPHB4eGFP/+hPSCs were induced to differentiate into CD34+endothelial progenitor cells,followed by functional investigation of EC-related factors in the specialization of endothelial progenitor cells into AECs and VECs.The results of flow cytometry analysis showed that both noradrenaline(NE)and vascular endothelial growth factor(VEGF)could promote the formation of mCherry+AECs.The proportion of AECs was up to 90%post the combinational treatment of NE and VEGF for 6 days,while the proportion of eGFP+VECs was significantly reduced.Moreover,the fibroblast growth factor(bFGF)significantly promoted the differentiation of VECs from hPSCs while inhibited the formation of AECs.We then evaluated the molecular characterization and function of these hPSC-derived-AECs and VECs both in vivo and in vitro.Compared with the eGFP+VECs,the mCherry+AECs showed significantly higher expression of AECs-specific marker molecules including CXCR4,NRP1 and DLL4,while the expression levels of VECs-specific markers such as COUP-TFII,NRP2 and EPHB4 were significantly reduced.In addition,the ability of tube formation and nitrous oxide synthesis was much better in mCherry+AECs than that in eGFP+VECs.Moreover,both AECs and VECs obtained in this study were able to form blood vessels in vivo.Therefore,by using the hPSC-based fluorescent reporter system,we successfully established the specific differentiation methods for AECs and VECs.The methodological progress in hPSC-derived AEC and VEC induction will contribute to the mechanism study of AEC and VEC specification,arteriovenous disease modeling,endothelial cell therapy and artificial blood vessel construction.