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RNA interference (RNAi) holds great promise as a tool to study the basic biology of stem cells or to direct differentiation in a specific manner. Barriers to achieving efficient and specific gene silencing in RNAi experiments include limitations in transfection efficiency and in the efficacy and specificity of RNAi silencing effectors. Here, we combine methods of efficient lipid‐mediated delivery with chemically modified RNAi compounds to silence genes related to pluripotency, in order to direct differentiation of mouse embryonic stem cells. After transfection of embryonic stem cells with OCT4‐ or Nanog‐targeted RNAi compounds, levels of OCT4 or Nanog transcript and protein were reduced accordingly. Reduction in OCT4 expression correlated with induction of trophectoderm genes  Cdx2 ,  Hand1 , and  PL‐1 , with formation of cells with trophoblast giant cell phenotype after 6 days. Reduction in Nanog expression correlated with induction of extraembryonic endoderm genes  GATA4 ,  GATA6 , and  laminin B1 , with subsequent generation of groups of cells with parietal endoderm phenotype. Our results indicate that transient inhibition of OCT4 or Nanog by RNAi compounds is sufficient to induce differentiation toward extraembryonic lineages, which supports the model that these transcription factors function in a dose‐dependent manner to influence cell fate.

Differentiation of Mouse Embryonic Stem Cells after RNA Interference‐Mediated Silencing of OCT4 and Nanog