Author : Anil Khuttan, Kapil Dev, Birbal Singh, Dheeraj Mohania, Manoj Kumar, Vinod Verma,Anal Kant Jha and Sanjeev K. Gautam

The current study was carried out to isolate, culture, characterize and cryopreserve the amniotic fluid stem (AFS) cells in sheep. Putative AFS cells were cultured without feeder cells, in DMEM containing 10% FBS, 1% penicillin/ streptomycin and 1% L-glutamine in 5% CO2 in humidified air at 37±0.5˚C. After 6 days of culture different morphologies of cells were observed. Most of the cells started anchorage-dependent growth after day 6 of the culture. In order to check their pluripotency properties when the cells were subjected to RT-PCR a strong positive expression of Oct-4, Nanog and Sox2 was observed. Using species-specific primers, a PCR amplicon of 290, 501 and 362 bp was observed for Oct-4, Nanog and Sox-2 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 this species.

Keywords: Sheep, amniotic fluid, stem cells, characterization and RT-PCR.