We have used RNA interference (RNAi) to downregulate β2-microglobulin and Oct4 in human embryonal carci- noma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulat- ing specific gene activity in these human stem cells. The knockdown of Oct4 but not 2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferenti- ated stem cell state, and that differentiation to trophecto- derm occurs in its absence. NTERA2 hEC cells also dif- ferentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

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