Heterogeneous amniotic fluid contains various cell types. The aim of this study was to characterize and differentiate some of the key stemness attributes of the amniotic fluid-derived cells in buffalo (Bubalus bubalis). The amniotic fluid (AF) cells were cultured without feeder cells, in DMEM containing 15% FBS, 1% non-essential amino acids, 1% penicillin/streptomycin/ampicillin, 1% vitamin solution, and 1% l-glutamine in 5% CO(2) in humidified air at 38.5±0.5 °C. After 6 days of culture different types of cells viz., star shaped (62.7%), spherical without nucleus (1.9%), spherical with nucleus (26.4%), pentagonal (0.4%), and free floating/rounded cells (8.3%) were observed. Most of the cells started anchorage-dependent growth after day 7 of the culture. Expression of alkaline phosphatase (AP) and Oct-4, Nestin and FGF-5 were observed from the AF cells at different passages. Using species-specific primers, a PCR amplicon of 200, 296 and 210 bp were observed for Oct-4, Nestin and FGF-5, respectively. The cells were found to have a normal karyotype at different passages. These results may contribute towards establishing non-embryonic pluripotent stem cells for various therapeutic and reproductive biotechnological applications in the species.
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