Research For Validation Of Maintenance And Differentiation Potential In Human Trophoblast Stem Cells And Roles Of TEAD4,CDX2,GATA3 In Human Trophoblast Differentiation

Normal placental development is indispensable for normal pregnancy.And in the whole developing process from trophectoderm to mature trophoblast,mistakes in any stage can exert significant influence on pregnancy.Therefore,appropriate cell models are powerful tools for us to elucidate normal and abnormal regulation mechanisms of key gene expression in trophoblast development,and seek inspiration for diagnosis and treatment to pregnancy-relevant complications.And in our experiments,establishment of such a model,named human trophoblast stem cell model(hTSCs model)can hopefully bring about more advantages in the placenta-related research field.It is reported that human trophoblast stem cell model(hTSCs model)has been recently established in Japan,the study on which is,however,still a blank field in our country.Therefore,utilizing the generated human trophoblast stem cell lineages,we carry out a research for validation of maintenance and differentiation potential in human trophoblast stem cells.Specifically,using one of the TSC lineages,named hTSblast,we have developed a specialized cell culture system to maintain its self-renewal and then confirm its identification as human trophoblast stem cells.And two specific steps in the identification process have been organized,including identification of human trophoblast cells,and validation of stem cell characteristics about self-renewal and differentiation potential.In the first step,hTSblast was identified as human trophoblast cells according to the following principles:1).Expression of trophoblast markers such as KRT7,GATA3,TFAP2C,etc;2).Expression of 4 miRNAs from C19MC.In the second step,it has also been validated that hTSblast possesses certain cell proliferation capability as well as differentiation potential.That is,we have selectively induced hTSblast hTSCs to differentiate into extravillous trophoblast(EVT)or syncytiotrophoblast(STB)and then confirmed the identification of the mature trophoblast cells based on evidence about their characteristics including distinct cell morphology,downregulated expression of TS markers and upregulated expression of differentiation markers,involving HLA-G/MMP2 in EVT,and CGB/CGA in STB.In another part of our whole experiments,research is focused on roles of TEAD4,CDX2 and GATA3 in the trophoblast differentiation process.Here we adopt the hESCs trophoblast differentiation model(BAP model).After generation of TEAD4/CDX2/GATA3 knockout human embryonic stem cell lineages through CRISPR/Cas9 technology,BAP differentiation method is performed to compare the effectiveness of trophoblast differentiation among H1-TEAD4/CDX2/GATA3-/-hESCs and H1 hESCs.And it is concluded from these results that,TEAD4/CDX2 deletion can lead to attenuated trophoblast differentiation,while GATA3 deletion appears to have no impact on differentiation potential in H1 hESCs,and overexpression of TEAD4,CDX2 or GATA3 alone in H1 TEAD4-/-hESCs is sufficient to correct the defect caused by TEAD4 deletion in transition from human embryonic stem cells to trophoblast.Finally,in absence of BMP4,overexpression of GATA3 in H1 hESCs result in spontaneous trophoblast differentiation,while H1 hESCs with overexpression of CDX2 still maintain embryonic stem cell state.From these results,it can be inferred that TEAD4-CDX2 axis is necessary for human trophoblast differentiation while GATA3 alone is dispensable,and between GATA3 and TEDA4-CDX2 axis exists functional redundancy,exaggerated especially in TEAD4 deletion condition.