Background: Intrinsic factors and extrinsic signals which control unlimited self-renewal and developmental pluripotency in embryonic stem cells (ESCs) have been extensively investigated. However, a much smaller number of factors involved in extra-embryonic trophoblast differentiation from ESCs have been studied. In this study, we investigated the role of the single-stranded DNA binding protein, Ssbp3, for the induction of trophoblast-like differentiation from mouse ESCs.
Methods: Gain- and loss-of-function experiments were carried out through overexpression or knockdown of Ssbp3 in mouse ESCs under self-renewal culture conditions. Expression levels of pluripotency and lineage markers were detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses. The global gene expression profile in Ssbp3-overexpressing cells was determined by affymetrix microarray. Gene ontology and pathway terms were analyzed and further validated by qRT-PCR and Western blotting. The methylation status of the Elf5 promoter in Ssbp3-overexpressing cells was detected by bisulfite sequencing. The trophoblast-like phenotype induced by Ssbp3 was also evaluated by teratoma formation and early embryo injection assays.
Results: Forced expression of Ssbp3 in mouse ESCs upregulated expression levels of lineage-associated genes, with trophoblast cell markers being the highest. In contrast, depletion of Ssbp3 attenuated the expression of trophoblast lineage marker genes induced by downregulation of Oct4 or treatment with BMP4 and bFGF in ESCs. Interestingly, global gene expression profiling analysis indicated that Ssbp3 overexpression did not significantly alter the transcript levels of pluripotency-associated transcription factors. Instead, Ssbp3 promoted the expression of early trophectoderm transcription factors such as Cdx2 and activated MAPK/Erk1/2 and TGF-β pathways. Furthermore, overexpression of Ssbp3 reduced the methylation level of the Elf5 promoter and promoted the generation of teratomas with internal hemorrhage, indicative of the presence of trophoblast cells.
Conclusions: This study identifies Ssbp3, a single-stranded DNA binding protein, as a regulator for mouse ESCs to differentiate into trophoblast-like cells. This finding is helpful to understand the regulatory networks for ESC differentiation into extra-embryonic lineages.
Keywords: Differentiation; Mouse embryonic stem cells; Ssbp3; Trophoblast.