HUMAN EMBRYONIC STEM CELLS STABLY OVER-EXPRESSING EGFP MAINTAIN PLURIPOTENCY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To prepare genetically marked human embryonic stem cells with a visual marker. The availability of human ES cells with a readily evaluated genetic marker such as green fluorescent protein will facilitate a number of experimental opportunities. We replaced the CMV and SV40 promoters in pCDNA3.1 (Invitrogen, Carlsbad, CA) with 2 EF-1alpha promoters to prepare the YPL2 vector with mammalian promoters for transgene expression in human ES cells. EGFP cDNA was inserted under the control of the first EF-1 alpha promoter to construct plasmid YPL2-EGFP. The second EF1 alpha promoter was upstream of the neomycin resistance gene. H1 or H9 human embryonic stem (HES) cells were transfected with YPL2-EGFP using Fugene 6. Following 100 ug/ml neomycin selection for 12 days, individual colonies demonstrating stable EGFP expression were observed. After 3 months of passage under neomycin selection, the cells continued to maintain typical HES cell morphology. Immunostaining demonstrated maintenance of Oct-3/4 expression which was indistinguishable from untransfected HES cells. Adding 10 ng/ml BMP4 to the cells provoked differentiation to trophoblasts, with dramatically increased hCG secretion and morphological changes but no loss of EGFP expression. After 3 months of passage the cells showed no change in EGFP expression as determined by FACS analysis. Following injection of EGFP-HES cells into scid mice, there was robust formation of teratomas that demonstrated a broad range of morphological pluripotency. EGFP expression was widespread in differentiated cells of these teratomas. The results obtained with this plasmid were more consistent than with lentiviral vectors and IRES elements to select for stable transfectants. HES cells carrying EGFP (or other readily identified visual or metabolic markers) will be useful in many other areas of research such as differentiation, migration and morphogenesis. This research used WNPRC Animal Services and Research Services, including federally approved human ES cell lines.