Isolation, culture, and induced multiple differentiation of Mongolian sheep adipose-derived mesenchymal stem cells

Adipose-derived mesenchymal stem cells (ADSC) are adult pluripotent cells and important resources for cell-based therapies of animals. There are presently different kinds of somatic cells used as donor cells for clone successfully. However, studies on somatic cell nuclear transplantation (SCNT) using ADSC as donor cells from Mongolian sheep have not been reported up to now. This study tested optimal methods of isolating, purifying, and proliferating Mongolian sheep ADSC, and determine their multiple differentiation potentiality. Adipose tissue was removed from approximately 2-year-old sheep and ADSC were harvested by pancreatic enzyme decomposition and adherent culture method. The growth curves of the Passages 1, 5, and 10 cultures were plotted and the exponential growth was determined as a population doubling time of 34.1 h. The expression of OCT4, SOX2, and NANOG genes were increased at Passage 3 (P3) as seen by reverse transcription polymerase chain reaction (RT-PCR) analysis. ADSC from Passage 3 were induced to undergo neurogenesis and form cardiomyocytes and pancreatic islet-like cells under inductive environments in vitro. The differentiation properties of cardiomyocytes and islet-like cells were confirmed by histological staining with toluidine blue, periodic acid-Schiff, and dithizone. The expression of specific genes in these cells were also detected by RT-PCR. Our study results confirm that isolated cells were indeed ADSC and may provide valuable materials for somatic cell clone and transgenic research.