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RAPID COMMUNICATION |
a The Centre for Stem Cell Biology and the Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, U.K.;
b Axordia Ltd, Western Bank, Sheffield, U.K.;
c Section of Reproductive and Developmental Medicine, University of Sheffield, Royal Hallamshire Hospital, Jessop Wing, Sheffield, U.K.
Key Words. Human embryonic stem cells • Embryonal carcinoma cells • RNA interference Oct4 • ß-2-microglobulin • GFP • Differentiation • Gene expression
Correspondence: P. W. Andrews,B.Sc., D.Phil., M.B.A., The Centre for Stem Cell Biology and the Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, U.K. Telephone: +44 (0)114-222-4173; Fax: +44 (0)114-222-2399; e-mail: p.w.andrews{at}sheffield.ac.uk
We have used RNA interference (RNAi) to downregulate ß2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not ß2-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 undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.
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