Present study examined the effect of mitomycin-C (MMC) treatment on expression profi le of pluripotency genes (Oct-4, Sox-2 and Nanog) in buffalo fetal fi broblasts (BFFs) and Wharton’s jelly (BWJ) stem cells, generally used as a feeder cell support to grow the pluripotent stem cells. In a time dependent study, a variable response in relative mRNA expression of pluripotency genes was observed, expression of Oct-4 in BFF declined immediately post MMC inactivation but a signifi cant elevation (P<0.05) was noticed later on. The relative mRNA expression remained unchanged in BWJ, up to 24 h post MMC treatment and thereafter, it increased signifi cantly (P<0.05). A similar trend for the expression pattern of Sox-2 and Nanog was observed in both the cell types. MMC inactivation caused an upregulation in the expression of Sox-2 and Nanog in BFF while it remained unchanged (P>0.05) in BWJ. Results of this study suggests that MMC inactivation of the cells used as feeder support modulates the expression profi le of pluripotency genes and this alteration in gene expression pattern is variable in different cell types.