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Human embryonic stem (hES) cels are capable of diferentiating into pluralistic cel types, how-ever, spontaneous diferentiation generaly gives rise to a limited number of specific diferentiated cel types and a large degree of cell hetero-geneity. In an effort to increase the eficiency of specified hES cell diferentiation, we per-formed a series of transient transfection of hES cels with EGFP expresion vectors driven by diferent promoter systems, including human cel-(hEF1α), human cytomegalo-virus, and chicken β-actin. Al these promoters were found to lead reporter gene expresion in undiferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable ex-pression of the transgene with either chemical or electroporation methods. In an attempt to in-crease transfection eficiency and obtain stable transgene expression, diferentiated hES cells ex-pressing both mesodermal and ectodermal mar-ferentiated hES cels were electroporated with a hEF1α promoter-driven EGFP or human noggin expresion vector. Using RT-PCR, immunocyto-chemistry and fluorescence microscopy, the dif-ferentiated hES cels transfected with foreign genes were confirmed to retain stable gene and protein expresion during prolonged culture. These results may provide a new tol for in-troducing exogenous genes readily into hES cels, thereby facilitating more directed diferentiation into specific and homogenous cel populations.

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