Role And Mechanism Of TEAD4 In Early Human Trophoblast Development Based On Human Embryonic Stem Cells Towards Trophoblasts Differentiation Model

During embryonic development,trophoblast derived from trophectoderm of blastocyst is vital for embryo implantation,placenta formation,and maintaining healthy pregnancy.Early trophoblast differentiation defects may not only lead to implantation failure of artificial assisted reproductive procedure but also miscarriage,intrauterine growth restriction,preeclampsia or other placenta derived diseases.Recent studies have even found that placentation defects could lead to fetal heart and brain abnormalities through intrauterine programming.However,research on trophoblast development is quite limited due to the lack of effective early trophoblastic differentiation models.While human embryonic stem cells towards trophoblasts differentiation model has unique advantages.Human embryonic stem cells(hESCs)are a group of cells with unlimited proliferation and multilineage differentiation potential,that can be induced to differentiate into various cell types in vitro.hESCs can also be induced towards trophoblast lineage under culture of bone morphogenetic protein 4(BMP4),which provide a research model for study key factors in regulating human trophoblast development.TEA domain family member 4(TEAD4)is a crucial transcription factor in mouse trophoblast differentiation.In early murine embryonic development,TEAD4 initiates the differentiation of trophectoderm and regulates the expression of a series of trophoblast transcription factors.However,the function of TEAD4 in the process of human trophoblast development has not been clarified yet.In recent reports,there is still controversy about whether TEAD4 is involved in regulating early human trophoblast differentiation.In this study,we used the human embryonic stem cells towards trophoblasts differentiation model by E6+BAP(BMP4/A83-01/PD 173074)treatment as previously reported to study the function of TEAD4 in early human trophoblast differentiation.First,we generated H1-TEAD4-/-cell lines using CRISPR/Cas9,and tested the pluripotency.The results showed no alteration in pluripotency,based on pluripotent markers expression and teratoma formation.And then,when differentiated towards trophectoderm lineage under the condition of E6+BAP,H1-TEAD4-/-cell showed a significant decrease in trophectoderm specific gene expression and hormone secretion,compared with wild-type cells.Furthermore,overexpression of TEAD4 in H1-TEAD4/-cells rescued the expression level of TEAD4.After trophectoderm differentiation,H1TEAD4-/-+TEAD4 cells showed significant increase in trophectoderm specific gene expression and hormone secretion compared with H1-TEAD4" cells,while no significant difference with wild-type cells.At last,we generated H1-TEAD4-/-+ GATA3 by over-expression of GATA3 in H1-TEAD4-/-cells.H1-TEAD4-/-+GATA3 cells also showed significant increase in trophectoderm specific gene expression and hormone secretion compared with H1-TEAD4-/-cells,while no significant difference with wildtype cells.Collectively,our data indicated that TEAD4 cooperate with GATA3 to regulate early human trophoblast development based on human embryonic towards trophoblast differentiation model.These results will provide evidence to gain a better understanding of trophoblast cells differentiation regualatory networks.