Isolation, culturing and characterization of feeder-independent amniotic fluid stem cells in buffalo (Bubalus bubalis)

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% CO2 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.