Cultured buffalo umbilical cord matrix cells exhibit characteristics of multipotent mesenchymal stem cells

Recent findings have demonstrated umbilical cord, previously considered as a biomedical waste, as a source of stem cells with promising therapeutic applications in human as well as livestock species. The present study was carried out to isolate the umbilical cord matrix cells and culture for a prolonged period, cryopreserve these cells and test their post-thaw viability, characterize these cells for expression of stem cell markers and differentiation potential in vitro. The intact umbilical cord was taken out of the amniotic sac of a fetus and then incised longitudinally to remove umbilical vessels. Wharton's jelly containing tissue was diced into small pieces and placed in tiny drops of re-calcified buffalo plasma for establishing their primary culture. Confluent primary culture was trypsinized and passaged with a split ratio of 1:2 for multiplication of cells. Cryopreservation of cells was performed at three different passages in cryopreservation medium containing 15%, 20% and 25% fetal bovine serum (FBS). A significant increase in post-thaw viability was observed in cells cryopreserved in freezing medium with higher concentration of FBS. After re-culturing, frozen-thawed cells started adhering, and spike formation occurred within 4-6 h with similar morphology to their parent representative cultures. The normal karyotype and positive expression of alkaline phosphatase and pluripotency genes OCT4, NANOG and SOX2 were observed at different passages of culture. When induced, these cells differentiated into adipogenic and osteogenic cells as confirmed by oil red O and alizarin red stains, respectively. This study indicates that buffalo umbilical cord matrix cells have stemness properties with mesenchymal lineage restricted differentiation and limited proliferation potential in vitro.