Since the birth of the first cloned sheep was reported, fibroblasts are commonly used as donor cells for nuclear transfer. However, in some species there is a higher incidence of abnormal fetuses, still births, and neonatal deaths as a consequence of reprogramming disorders including DNA methylation and histone acetylation. Cloned embryos with mesenchymal stem cells (MSC) have shown higher developmental ability compared to fibroblast, suggesting that undifferentiated genome can required ease reprogramming. Because MSC are relatively difficult to collect from bone marrow, skin is an alternative source of the donor cells. However, molecular and functional analyses remain uncertain between MSC derived from bone marrow and skin stem cells isolated from ear. The present study compared the expression of early transcription factors (Oct-4, Nanog and Sox-2), and differentiation capability to osteocytes, adipocytes and chondrocytes of MSC isolated from bone marrow and skin-derived putative stem cells from miniature pig. Bone marrow was isolated by Ficoll density gradient method, and skin separated from epidermis and dermis was diced into 2-mm diameter explants, and attached to tissue culture dishes. Cells were then cultured in DMEM/F12 supplemented with 10% FBS, 10 ng mL–1 bFGF, 10 ng mL–1 EGF at 38.5°C, in a humidified atmosphere of 5% CO2 in air. Expression of Oct-4, Nanog, Sox2 was analysed by RT-PCR. Osteogenic and adipogenic differentiation were induced following the protocols described previously (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90; Mohana Kumar et al. 2007 Mol. Cells 24, 343–350) and compared by histological staining and RT-PCR. Osteocytes were defined by the formation of the mineral nodules of deposition of calcium by Von Kossa staining and differentiations into adipocytes and chondrocytes were identified by oil red O staining of lipid vacuoles and alcian blue of proteoglycan, respectively. Skin-derived MSC were revealed to similar mRNA expression of Oct-4, Nanog, Sox2 compared to bone marrow derived MSC. However, bone marrow derived MSC were higher mRNA expression about of osteocytic genes (osteoclacin and osteonectin), chondrocytic gene (collagen type) and adipocytic genes (aP2) than those of skin-derived MSC. In addition, bone marrow derived MSC were revealed greater deposition of calcium, proteoglycan, and lipid vacuoles than those of skin derived MSC by histological staining. The results of present study suggest that cells isolated from skin have lower potential than MSC isolated from bone marrow. However, skin-derived stem cells have properties of multi-lineage differentiation and can be obtained easily. These stem cells, therefore, can serve as easily accessible and expandable source possessing donor cells for cloning, potential animal model, and clinical applications.
This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.
Developmental Ability of Miniature Pig Embryos Cloned with Mesenchymal Stem Cells
