The role of let-7 in human trophoblast differentiation

Abstract

<p>First lineage segregation involves the formation of inner cell mass and trophectoderm. This process is crucial for proper embryo development and subsequent implantation. Because of the scarcity of human embryos for research, our current knowledge on the molecular events in lineage segregation mainly relies on mouse models. Our previous studies demonstrated the importance of a family of microRNA lethal-7 (let-7) in early mouse embryo development. We found over-expression of let-7 during cleavage stage led to lower blastocyst formation rate and embryo dormancy. More importantly, let-7 targeted and downregulated <em>Tead4</em>, an important trophectoderm marker. We hypothesize in this study that let-7 has a conserved role for modulating trophectoderm development in humans and mice. Here we utilized a novel human expanded potential stem cell derived from human embryos (hEPSC-em) as an <em>in-vitro</em> model for study. hEPSC-em was differentiated efficiently into early trophoblast lineage by administration of BMP4, A83-01 and PD173074 in the form of embryoid bodies (BAP-EB). Similar to the primed hESC, BAP-EB from hEPSC-em showed cystic structure at 48H post-differentiation with size and morphology reminiscent of human blastocysts. Moreover, single-cell RNA sequencing data revealed that the BAP-EB formed from hEPSC-em had high expressions of early trophoblast markers (<em>CDX2</em>, <em>GATA2</em> and <em>GATA3</em>). We detected that the expression of let-7 was significantly downregulated during BAP-EB formation. More importantly, forced expression of let-7 mimic significantly hindered the cystic structure formation of BAP-EB at 48H post-differentiation. We sought to identify the downstream target of let-7 during human early trophoblast development. <em>In-silico</em> analysis predicted <em>YAP1</em>, an important human trophectoderm effector, was downstream target of let-7. We further confirmed the downregulation of YAP1 protein upon let-7 overexpression in BAP-EB. Our results suggested that let-7 has a conserved role in regulating human and mouse trophectoderm and early trophoblast differentiation.</p>