Establishment Of Cynomolgus Macaque(Macaca Fascicularis) Trophoblast Stem Cells

The placenta forms a maternal-fetal junction that supports many physiological functions such as the supply of nutrition and the exchange of gases and wastes.Trophoblast stem cell is the precursor cell of placental differentiation,but molecular mechanisms regulating human trophoblast development remain poorly understood.Thus,establishing an in vitro culture model of human and non-human primate trophoblast stem cells is important for investigating the process of early placental development and trophoblast differentiation.In this study,we have established three trophoblast stem cell(TSC)lines from embryos of a cynomolgus monkey in vitro delayed culture,named cynomolgus monkey E15\E20\E19-TS cells.This cynomolgus monkey TS cells showed a trophoblastic gene expression profile: GATA3,TFAP2 C,CK7(KRT7)and TEAD4.Exhibited differentiation capacity towards extravillous trophoblast(EVT)cells and syncytiotrophoblast(ST)cells.Cynomolgus monkey TS cells differentiated into EVT expressed MMP2 and N-cadherin,which are related to the invasiveness of EVT.Two different culture methods ST-2D and ST-3D were used to differentiate TS cells into ST.GCM1 and E-cadherin immunofluorescence staining were used to identify the differentiated cells.The results showed that the differentiated cells of ST-2D were partially multinucleated,and the induced differentiation to the ST medium could change the cell membrane and promote the fusion of some cells.Differentiated cells all highly expressed genes related to trophoblast differentiated cells:SDC1,ITGA5,MMP2,GCM1 and CBG.Especially,EVT and ST-3D cells can secrete progesterone.In the xenogeneic chimera assay,the cynomolgus monkey TS cells only contributed to trophectoderm(TE)development in the chimeric embryos of E4.5 and E6.4.