Establishment And Identification Of Trophoblast Stem Cell (TSC) Lines From Human Embryonic Stem Cell(hESC)

Objective To establish and identify the trophoblast stem cell lines(TSC)from human embryonic stem cell(hESC)after the hESC was induced to differentiate into trophoblast stem cell-like cells by BMP4.Methods Single cell H9 was induced to differentiate into trophoblast stem cell-like cells by 10ng/ml BMP4 in the condition of feeder free layer.EGF,activator of Wnt,inhibitor of TGF??HDAC and ROCK were added into culture medium to maintained cell proliferation and passage.The expression of CTB markers(CDH1,EGFR,etc.)were detected and the cell proliferation curve was mapped.Also,trophoblast stem cells were differentiated into EVT and STB respectively by NRG1+Matrigel or Forskolin.Results After four days of hESC differentiation induced by BMP4,the cells presented a flat TS-like morphology under microscopy.At this time,the positive cell rates of CDH1,EGFR and KRT7 markers were 81.00%,58.00% and 75.67%,respectively,indicating the high efficiency of this differentiation system.The cell then adapted well in TS medium.The positive ratio of CDH1 and KRT7 to the third generation were 88.08% and 91.38%,respectively.Also,TSP3 expressed GATA3,P63,TFAP2 C,CDX2 and TEAD4,and did not express HLA-G,HCG and OCT4,and could differentiate into both EVT and STB-like cell at the same time,proving that high-purity TS cells were obtained.However,with the extension of culture days,the positive rate of TSP5 decreased to 12.5%,and the positive rate of CDH1 and KRT7 also decreased,but more than 60% of the cells still expressed the markers.After that,the cell proliferation ability was significantly reduced in TSP7,and the expression of CDH1 and KRT7 could hardly be detected.Conclusion The trophoblast stem cell-like cells obtained from BMP4-induced hESC can be proliferated in TS medium but with limited proliferation capacity,and the culture systerm of this differentiation model still needs to be further optimized.At the same time,we need to further identify the stage of trophoblast cell.