DIFFERENTIATION OF PRIMORDIAL GERM LIKE CELLS FROM HUMAN EMBRYONIC STEM CELLS

Human Embryonic stem cells (hESC) offers scientist a new tool to better understand human germ cell specification and signaling pathways associated with germ cell development. Two markers often associated with germ cell differentiation, vasa and SSEA-1 are not expressed in undifferentiated human embryonic stem cells. Therefore monitoring the expression of these markers during differentiation may provide information on germ cell formation. Also, basic fibroblast growth factor (β-FGF) has been shown to increase the proportion of mouse embryonic stem cells that differentiate toward the germ cell lineage. The objectives of this study were to determine the effect of time in culture and β-FGF on expression of SSEA-1 and Vasa markers in differentiating populations of hESCs. The experiment was a 2 by 3 factorial with time points at day 3, 10 and 30 with and without β-FGF. H9 hESCs were maintained on mitomycin C inactivated mouse embryonic feeder (MEF) layers in DMEM/F12, 20% Knockout Serum Replacer, 2 mM L-glutamine, 0.1 mM MEM non-essential amino acids, 50 U/ml penicillin, 50 μg/ml streptomycin (all from Gibco/Invitrogen), 0.1 mM βME (Sigma), and 4 ng/ml βFGF (Sigma). Undifferentiated hESC were plated as clumps of manually disassociated colonies onto 4 well chamber slides that were previously plated with inactivated MEFs. The cells were cultured in the presence or absence βFGF for the appropriate amount of time (3, 10 and 30 days). At the end of these three time points, cells were fixed and immunocytochemical analysis was performed for SSEA-1 and Vasa epitopes. We found that there was a subpopulation of differentiating cells that expressed the germ cell markers SSEA-1 and Vasa. There was a trending increase in SSEA-1 expression from day 3 to day 10 and a statistically significant decrease in SSEA-1 expression on day 30, but there was no effect of β-FGF during this time. There was no affect of time and β-FGF on vasa expression however we observed a clear trend toward increased vasa expression after prolonged culture in the presence of β- FGF. Importantly, vasa expression was localized in the nucleolus. Cellular localization of vasa expression has not been previously determined in any species and this study provides new insight into the possible role for vasa expression in the differentiation human germ cells. (poster)