Amniotic mesenchymal stem cells (AMSCs) from livestock are valuable resources for animal reproduction and veterinary therapeutic. The purpose of this study is to explore a suitable way to isolate and culture the buffalo AMSCs (bAMSCs), and to identify their biological characteristics. Digestion with a combination of trypsin-EDTA and collagenase type I could obtain pure bAMSCs more effectively than trypsin-EDTA or collagenase type I alone. bAMSCs could proliferate steadily in vitro culture and exhibited fibroblastic-like morphology in vortex-shaped colony. bAMSCs were positive for MSC-specific markers CD44, CD90, CD105, CD73, β-integrin (CD29) and CD166, and pluripotent markers OCT4, SOX2, NANOG, REX-1, SSEA-1, SSEA-4 and TRA-1-81, but negative for hematopoietic markers CD34, CD45 and epithelial cells specific marker Cytokeratin 18. In addition, bAMSCs were capable of differentiating into adipogenic, osteogenic, chondrogenic and neural lineages, with expression of FABP4, Ost, ACAN, COL2A1, Nestin and β III-tubulin. Glycogen synthase kinase 3 inhibitor: kenpaullone promoted bAMSCs to differentiate into neural lineage. This study provides an effective protocol to obtain and characterize bAMSCs, which have proven useful as a cell resource for buffalo cell reprogramming studies and transgenic animal production.

Adipose tissue‐derived mesenchymal stem cells (ASCs) from livestock are valuable resources for animal reproduction and veterinary therapeutics. Previous studies have shown that hypoxic conditions were beneficial in maintaining the physiological activities of ASCs. However, the effects of hypoxia on buffalo ASCs (bASCs) remain unclear. In this study, the effects of hypoxia on proliferation, stemness, and reprogramming into induced pluripotent stem cells (iPSCs) of bASCs were examined. The results showed that the hypoxic culture conditions (5% oxygen) enhanced the proliferation and colony formation of bASCs. The expression levels of proliferation‐related genes, and secretion of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were significantly enhanced in hypoxia. Hypoxic culture conditions activated hypoxia‐inducible factor‐1α (HIF‐1α), thereby contributing to the secretion of bFGF and VEGF, which in turn enhanced the expression of HIF‐1α and promoted the proliferation of bASCs. Furthermore, in hypoxic culture conditions, bASCs exhibited the main characteristics of mesenchymal stem cells, and the expression levels of the pluripotent markers OCT4, NANOG, C‐MYC, and the differentiation capacity of bASCs were significantly enhanced. Finally, bASCs were more efficiently and easily reprogrammed into iPSCs in hypoxic culture conditions and these iPSCs exhibited some characteristics of naïve pluripotent stem cells. These findings provide the theoretical guidance for elucidating the detailed mechanism of hypoxia on physiological activities of bASCs including proliferation, stemness maintenance, and reprogramming.

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