author(s)
A Palanisammi, S Satheshkumar, M Vinoth, A Stalin Vedamanickam and K Kumanan

abstract
The objective of present study was to culture and characterize the buffalo embryonic stem cell-like cells (BESC) for their pluripotency by analysing Oct-4 and TRA-1-81 markers. Blastomeres from two, four, eight, sixteen, morula and blastocyst of in-vitro produced buffalo preimplantation embryos were derived and were seeded on mitomycin inactivated buffalo foetal fibroblasts feeder layer and cultured. The cultured blastomeres were monitored colony formation. The colonies exhibiting typical morphological features of BESC were subcultured. Marker expressions were tested on passages 1, 2, 3 and 4 by RT-PCR, immune-cytochemistry and flowcytometric analysis. RT-PCR study revealed that expression of Oct-4 progressively declined with subsequent passages. The BESC strongly reacted with TRA-1-81 antibodies. In flowcytometry, the cells expressing TRA-1-81 in the first passage and second passage were 98.03±0.03 per cent and 97.13±0.07 per cent respectively. However, in the third and fourth passages the expression levels were reduced to 12.11±0.04 and 4.21±0.09 per cent respectively. Thus the study proved that cultured BESC had pluripotency characters and from third passage they started differentiation process.