New insights for Ets2 function in trophoblast using lentivirus-mediated gene knockdown in trophoblast stem cells

Mouse trophoblast stem (TS) cells represent a unique in vitro system that provides an unlimited supply of TS cells for the study of trophoblast differentiation and TS cell self-renewal. Although the mouse transcription factor Ets2 is required for TS cell self-renewal, its role in this and in TS cell differentiation has not been explored fully, partly due to the early lethality of Ets2 null mice. To address this, we developed a novel lentivirus-based system that resulted in efficient Ets2 knockdown in the overwhelming majority of TS cells. This system enables functional studies in TS cells, especially for genes required for TS cell self-renewal because TS cell derivation using gene-knockout embryos for such genes depends on TS cell self-renewal. Using morphological/morphometric criteria and gene expression analysis, we show that the requirement for Ets2 in self-renewal of TS cells cultured in 'stem cell medium' (SCM) involves maintenance of the expression of genes that inhibit TS cell differentiation in SCM, such as Cdx2 and Esrrb, and preservation of the undifferentiated TS cell morphology. During TS cell differentiation caused by Cdx2/Esrrb downregulation, due to either Ets2 knockdown in SCM or culture in differentiation medium (DM), Ets2 is also required for the promotion of trophoblast giant cell (TGC) and junctional zone trophoblast (JZT) differentiation. This TGC differentiation involves Ets2-dependent expression of Hand1, a gene required for the differentiation of all TGC types. This study uncovers new roles for Ets2 in TS cell self-renewal and differentiation and demonstrates the usefulness of this lentivirus system for gene function studies in TS cells.