DIFFERENTIAL TROPHOBLAST FORMATION FROM RHESUS EMBYONIC STEM CELLS

University Of Wisconsin-Madison, Madison WI

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Abstract

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.[unreadable] [unreadable] The differentiation potential of nonhuman primate embryonic stem cell (ESC)-like lines has been evaluated through [unreadable] spontaneous differentation in vitro, in teratomas, and with specific paradigms for neural or hematopoetic lineage [unreadable] differentiation. We had previously reported that the rhesus ESC line R278.5 initiated chorionic gonadotropin (CG) gene [unreadable] expression with spontaneous differentiation in vitro. We have further evaluated trophoblast differentiation in this line [unreadable] and another widely used rhesus ESC line, R366.4 cells, in embryoid body formation, and in treatment with BMP-4, as [unreadable] well as a range of other activin/TGF-beta and FGF family members. None of the factors tested induced CG secretion or [unreadable] morphological differentiation resembling trophoblast formation seen in the H1 and other human ESC lines treated with [unreadable] BMP4. However, R278.5 cells were able to consistently differentiate to trophoblasts during embryoid body formation, as [unreadable] evidenced by expression of CG, and outgrowths from rhesus embryoid bodies initiated expression of the trophoblast-[unreadable] specific MHC Class I molecule Mamu-AG. Under these conditions, however, there was no evidence of trophoblast [unreadable] differentiation in R366.4 ESC.[unreadable] [unreadable] To gain further insight into the differences that potentially underly these remarkably diverse differentiation potentials, [unreadable] we employed Affymetrix rhesus monkey microarrays using RNA from 5 independent cultures each of R278.5 and [unreadable] R366.4 ESC of similar passage number. Of the 52,866 probe sets measured, 32% were significantly differentially [unreadable] expressed, 61 probe sets had a 15-fold difference in the level of expression between the two lines, and 1,672 probe [unreadable] sets differed by 3-fold. The genes that were more highly expressed (all 10-fold) in R366.4 cells included stem cell [unreadable] related genes, e.g., SALL3, SALL4, SOX2, PP1R1A, GABARB3, Oct4, and nanog. We conclude that there is a differential [unreadable] responsiveness of rhesus ESC lines to paradigms that reliably promote human ESC differentiation to the trophoblast [unreadable] lineage. Differential gene expression as revealed by microarray analysis may provide insights into the properties that [unreadable] direct differentiation associated with early embryonic developmental decisions. This research used WNPRC Stem Cell [unreadable] Resources.