Isolation, Culture, Differentiation, and Nuclear Reprogramming of Mongolian Sheep Fetal Bone Marrow-Derived Mesenchymal Stem Cells.

We have characterized the differentiation potentiality and the developmental potential of cloned embryos of fetal bone marrow mesenchymal stem cells (BMSCs) isolated from Mongolian sheep. BMSCs were harvested by centrifuging after the explants method and the mononuclear cells obtained were cultured. The isolated BMSCs were uniform, with a fibroblast-like spindle or stellate appearance, and we confirmed expression of OCT4, SOX2, and NANOG genes at passage 3 (P3) by RT-PCR. We measured the growth of the passage 1, 5, and 10 cultures and found exponential growth with a population doubling time of 29.7±0.05 h. We cultured the P3 BMSCs in vitro under inductive environments and were able to induce them to undergo neurogenesis and form cardiomyocytes and adipocytes. Donor cells at passages 3-4 were used for nuclear transfer (NT). We found the BMSCs could be expanded in vitro and used as nuclear donors for somatic cell nuclear transfer (SCNT). Thus, BMSCs are an attractive cell type for large-animal autologous studies and will be valuable material for somatic cell cloning and future transgenic research.