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 define the ability of rhesus embryonic stem cells to differentiate into trophblasts. Primate embryonic stem cell (ESC)-like lines were first isolated from in vivo-produced rhesus monkey blastocyst- stage embryos, however the differentiation potential of the available lines has not been extensively compared. We evaluated the capacity of rhesus (r) ESC lines R366.4, R278, R394.3, R420 and R456 for trophoblast differentiation with paradigms shown to direct differentiation to trophoblasts in human (h) ESC. Trophoblast differentiation was assayed by secretion of monkey chorionic gonadotropin (mCG) or steroid hormones in undifferentiated rESC treated with 1 to 100 ng/ml BMP4, in suspension EB cultures, or in EBs grown in 2- or 3- dimensional environments, and by the histological appearance of cells expressing cytokeratin and mCG. Unexpectedly, none of the paradigms were able to increase trophoblast differentiation in R366.4 rESC, although substantial outgrowths were seen in 3-dimensional Matrigel culture. Given these results, we prepared embryoid bodies (EBs) from R278, R394.3, R420 and R456. Strikingly, rESC line R278 exhibited different properties from the other cell lines in general. EBs 1 day after formation from R278 cells had a substantial proportion of cytokeratin-positive cells distributed throughout early (day 1-2) EBs, and with further culture (up to day 24) cytokeratin expression becomes restricted to the periphery of these structures, similar to that seen with hESC- derived EBs. In addition, whereas EBs from R278 cells showed substantial differentiation to trophoblasts as evidenced by the presence of CG-positive cells within 8 days of EB formation in suspension. CG expression was not seen in R366, R420 or R456 cells, but was occasionally seen in R394.3 cells. We propose that differences among rhesus ESC cell lines in their capacity for trophoblast differentiation may allow insight into understanding the developmental origins of primate embryonic stem cell lines.
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