Spontaneous differentiation of buffalo (Bubalus bubalis) embryonic stem cells towards germ cell lineage

Abstract

Buffalo ES cells were subjected to differentiation in 15% KoSR-and 15% FBS-based spontaneous differentiation media in static suspension cultures. The embryoid bodies (EBs) so formed, were analyzed for germline-specific gene expression on day 4, 8 and 14 in order to identify the optimum differentiation strategy. Immunocytochemical analysis was performed for detection of germ lineage specific markers in EBs differentiated under the identified optimum conditions. Global DNA methylation analysis was performed to assay methylation erasure in the optimized differentiation cultures. We observed a significantly (p<0.05) increased expression of all germ lineage genes like DAZL, VASA and PLZF (PGC-markers); SYCP3, PRM2, TNP1/2 and MLH1 (Meiotic markers); BOULE and TEKT1 (Spermatocyte-markers); GDF9 and ZP2/3 (Oocyte-markers) upon spontaneous differentiation in comparison to undifferentiated ES cells. EBs collected from FBSbased medium showed significantly (p<0.05) higher expression of early germ lineage genes (DAZL, TNP1, PRM2), while those from KoSR-based medium showed greater expression of meiotic and developmental genes (SYCP3, MLH1, TEKT1, GDF9 and ZP2). Immunocytochemistry revealed expression of c-Kit, Dazl, Vasa (PGC-markers); Sycp3, Mlh1, Protamine1 (Meiotic markers); Acrosin and Haprin (Spermatocyte-makers); and Gdf9 and Zp4 (Oocyte-markers) in day 14 EBs collected from both the cultures. However, only PGC-specific markers were detected in 14 day monolayer cultures differentiated in KoSR-based media. The levels of 5-methyl-2-deoxycytidine were not significantly (p<0.05) different between EBs collected from the two media formulations. However, EBs collected from KoSRbased medium showed lower concentration of 5-Methyl-2-deoxycytidine, indicating comparatively greater methylation erasure.