Derivation, Characterization and Differentiation of Buffalo (Bubalus bubalis) Amniotic Fluid Derived Stem Cells

Abstract

Amniotic fluid cells (AFCs) are obtained from amnion for pre-natal analysis and can be cultured in vitro. Heterogeneous amniotic fluid (AF) contains various cell types, and it is believed that some of these cells possess the stem cell properties. The aim of this study was to characterize these cells by phenotypical and genotypical means in buffalo. The differentiation potential of amniotic fluid stem (AFS) cells was carried out by converting these cells into neurons. The AFCs were cultured without feeder cells in DMEM containing 16% foetal bovine serum, 1% penicillin/streptomycin and 1%l-glutamine in 5% CO(2) at 38.5 ± 0.5 °C in a CO(2) incubator. 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 Oct-4, Sox-2, Nanog, alkaline phosphatase, 18s rRNA, stem cell factor, cyclin A, Nestin and FGF-5 was observed from the AFS cells in different passages with PCR amplicon of 314, 277, 317, 180, 162, 216, 421, 307 and 210 bp, respectively. During the differentiation step, at day 6, neuron-like cells could be clearly identified and confirmed with Nestin-specific RT-PCR. 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.

Effects of Prolonged in vitro Culture and Cryopreservation on Viability, DNA Fragmentation, Chromosome Stability and Ultrastructure of Bovine Cells from Amniotic Fluid and Umbilical Cord

“…Dev et al. () reported similar results, but with buffalo AFC cultures. These researchers also reported that after 6 days of culture, cells from the same amniotic fluid sample were found with different shapes: star‐shaped, spherical‐shaped with and without nucleus, pentagonal‐shaped and round‐shaped.…”

“…The use of cells from extra‐embryonic tissues, umbilical cord and amniotic fluid can lead to significant advances in cell therapy, animal production and conservation (Dev et al. ). Extra‐embryonic tissues that originate from the hypoblast and trophectoderm do not participate in gastrulation and are therefore less differentiated than adult somatic cells (Cremonesi et al.…”

“…In the first trimester of pregnancy, amniotic fluid is composed of cells with the potential to differentiate into multilineages, presenting high growth kinetics and expression of pluripotent markers (Dev et al. ). A small percentage of cells from amniotic fluid represent a subpopulation of stem cells that express Oct‐4, the main specific marker of stem cell pluripotency (De Coppi et al.…”

Buffalo (Bubalus bubalis) term amniotic-membrane-derived cells exhibited mesenchymal stem cells characteristics in vitro

“…Expression of these pluripotency marker genes has previously been reported in amnion-derived cells of various mammalian species including rat (Marcus et al 2008), canine (Park et al 2012), chicken (Gao et al 2012), buffalo ) and human (Miki et al 2005). The expression of these genes has also been reported positive in cells derived from umbilical cord of porcine, equine and buffalo (Carlin et al 2006;Cremonesi et al 2008;Singh et al 2013) and buffalo amniotic fluid (Yadav et al 2011;Dev et al 2012).…”

“…Chromosomal spreads at passage 5 and passage 10 revealed that these cells had normal number of chromosomes (2n=50) indicating no apparent changes in ploidy during culture. Previous studies also reported a stable karyotype in cells derived from early buffalo gestation placental tissues (Yadav et al 2011;Dev et al 2012;Mann et al 2013;Singh et al 2013) and term amnion in equine (Seo et al 2013).…”