Abstract: Abnormal placentation is one of the underlying causes of pregnancy failure such as miscarriages and pre-eclampsia. Substantial progress has been made in deriving trophoblasts to study the early placental development. Human expanded potential stem cells (hEPSC) represent an efficient cellular platform for studying the early differentiation events during human embryo development. By using a human Expanded Potential Stem Cell (hEPSC) line with expanded potentials to both embryonic and extraembryonic lineages, this study aimed at identifying compounds that modulated early human trophectoderm/trophoblast development. A high throughput drug screening was carried out using hEPSC with GATA2 reporter (hEPSC-GATA2). Among the drugs that induced trophoblast development, the effects of three TGFβ inhibitors including RepSox, IN-1130 and SB-525334 on trophoblast differentiation were validated and the minimal effective dose was selected for further study. Real time qPCR and western blotting demonstrated that RepSox-, IN-1130- and SB-525334 treatments significantly induced the mRNA and proteins expressions of trophoblast markers. Besides, ELISA method indicated that RepSox and IN-1130 alone -induced the secretion of MMP2, but not hCG level. Furthermore, the addition of RepSox, IN-1130 and SB-525334 increased the attachment rate of the treated trophoblastic spheroid onto endometrial epithelial cells. In conclusion, RepSox, IN-1130 and SB-525334 can alone induce trophoblast differentiation from hEPSC. The TGFβ inhibition is indispensable for acquiring the attachment potential of trophoblastic spheroids. The identified novel pharmacological drugs and the related cell signalling pathways can provide new information of the early trophoblast differentiation and for future drug development for placental diseases.
