The Single-stranded DNA Binding Protein Ssbp3 Promotes Trophoblast Differentiation Of Mouse Embryonic Stem Cells

Derived from the Inner cell mass(ICM) of E3.5 embryos, mouse embryonic stem cells(ES cells) are capable of unlimited self-renewal in vitro and differentiation into any mature cell types. Too many studies focus on ES cell self-renewal and developmental pluripotency in recent years. However, the mechanism how to convert ES cells to extra-embryonic lineages remains less studied. Here we show that, the single-stranded DNA binding protein Ssbp3 plays a critical role in promoting ES cell differentiation into the trophoblast lineage.Ssbp3 is up-regulated during ES cell differentiation and overexpression of Ssbp3 promotes differentiation of mouse ES cells. These results suggest that Ssbp3 is associated with mouse ES cell differentiation. Expression analysis of specific markers related to pluripotency, three germ layers and extra-embryonic lineages showed that trophoblast specific markers were dramatically up-regulated, but pluripotency associated markers were not affected. These results indicated that Ssbp3 does not function through repression of core transcription factors of self-renewal. Instead, it drives the expression of the differentiation-related genes. Moreover, depletion of Ssbp3 attenuates the ability of ES cells to differentiate into the trophoblast induced by down-regulation of Oct4 in mouse ES cells. Global transcriptional profiling reveals that Ssbp3 promotes the trophoblast differentiation by driving the transcriptional program of the trophoblast, remarkably inducing the expression of early trophoblast transcription factors, such as Cdx2, Elf5 and Gata3, and activating the trophoblast-related pathways, MAPK pathway and TGF-! pathway. In addition, Ssbp3 overexpressing ES cells are able to form mature trophoblast cell types in vivo differentiation environment. Importantly, when injected into 8-cell-stage embryos, ES cells with ectopic Ssbp3 mainly contributed to trophoblast tissues in vivo. Taken together, our results identify Ssbp3 as a critical inducer of trophoblast development. Moreover, our works will facilitate the study about the conversion of ES cells to trophoblast, and the mechanism of trophoblast development.