The availability of human embryonic stem (HES) cells with a readily evaluated genetic marker such as green fluorescent protein (GFP) could facilitate a number of experimental opportunities. We constructed a novel plasmid with two elongation factor-1alpha (EF-1alpha) promoters (YPL2) to obtain a vector with mammalian promoters for simultaneous transgene expression in HES cells. An enhanced green fluorescent protein (EGFP) cDNA was inserted under the control of the first EF-1alpha promoter to construct plasmid YPL2-EGFP. The second EF1-alpha promoter was upstream of the neomycin resistance gene. H1 HES cells were transfected with YPL2-EGFP using Fugene 6. Following 100 microg/ml neomycin selection, individual colonies demonstrating stable EGFP expression were observed. After 4 months of passage under neomycin selection, the cells continued to maintain typical HES cell morphology. Undifferentiated cells showed no change in EGFP expression as determined by FACS analysis. Immunostaining demonstrated maintenance of Oct-3/4 expression in undifferentiated H1EGFP cells that was indistinguishable from wild-type HES cells. Addition of 10 ng/ml bone morphogenic protein-4 (BMP-4) to the cells provoked morphological and functional differentiation to trophoblasts, but no loss of EGFP expression. Following injection of EGFP-HES cells into immunodeficient mice, there was robust formation of teratomas that demonstrated a broad range of morphological pluripotency with widespread EGFP expression. EGFP expression was also maintained in differentiating embryoid bodies formed from EGFP-HES cells. This report demonstrates that ES cells carrying EGFP will be useful in diverse areas of embryonic stem cell research.
The earliest stages of human development profoundly influence the success of pregnancy and maternal as well as fetal health. One of the most critical developmental processes is the specification of the trophoblast lineage and the formation of the placenta, yet this is the stage that is most difficult to study because of the general inability to do in vitro studies with human embryos or conduct in vivo clinical research during the initial weeks of pregnancy. Embryonic stem cells potentially provide a novel surrogate for the undifferentiated pluripotent cells of the preimplantation embryo, and current research indicates that human embryonic stem cells are capable of differentiation to the trophoblast lineage under the influence of diverse soluble, transcriptional, and environmental cues. This review summarizes current areas of research in these different approaches to the study of placental cell differentiation and suggests avenues of investigation that are both unique and promising in this area.
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