Isolation And Characterization Of Bovine Skin-derived Precursor Cells And Their Developmental Potential After Nuclear Transfer

The skin is a complex and highly regenerative organ that houses a number of different precursor and stem cell populations. Such stem cell populations include both follicular and inter follicular epidermal stem cells, dermal mesenchymal stem cells, various hair follicle stem cells, endothelial and haematopoetic stem cells. A novel stem cell population was described by the laboratoryof Freda Miller in 2001 that have subsequently been termed skin-derived precursor cells(SKPs). These cells were isolated and expanded by methodology normally used to culture CNS stem cells and bore a superficial resemblance to CNS neuro sphere culture. When cultured in appropriate media, SKPs can give rise to neural and mesodermal lineages: including neurons, glial, adipocytes and smooth muscle cells. SKPs also represent a highly accessible and potentially autologous source of adult precursors that can generate different functional cell types, therefore, SKPs are a predominant autologous donor source for stem cell therapy. SKPs have also been isolated from mouse, humans, and swine. However, bovine SKPs have yet to be described. The purpose of this study is to isolate the skin derived precursor cells and to study the efficiency of nuclear transfer as a donor cell.The main contents are as follows:1. Bovine SKPs were successfully isolated from bovine fetal skin. Immunofluorescence analysis revealed that the SKP spheres were consistently positive for pluripotent markers,namely, SOX2, NANOG, and OCT4. The SKP spheres also expressed the nestin marker for neural precursors, fibronectin protein produced by bone marrow mesenchymalstem cells, and vimentin.The gene expression levels of NGFR, SNAI2, PAX3, SOX9, SOX2, NANOG, and OCT4 were subjected to QRT-PCR analysis. The results revealed that SKPs could produce higher mRNA levels of NCSC specifier(NGFR, SNAI2, PAX3, and SOX9) and pluripotent stem cell markers(SOX2, NANOG, and OCT4). To confirm the undifferentiated state of SKPs, we tested the expression of the following lineage related genes: GFAP for glial cells and β-III-tubulinand MAP2 for neurons. β-III-tubulin, MAP2, and GFAP did not obtain any detectable band. Therefore, SKPs were in an undifferentiated state. Immunocytochemicalanalysis revealed that the bovine SKPs generated numerous morphologically complex neurons that were positive for β-III-tubulinand GFAP. For glial differentiation, the expression of GFAP was observed. For neuronal differentiation, β-III-tubulin-positive subpopulation was present after differentiation was completed. This finding indicated the neuronal potency of SKPs. In the same differentiation cultures for neurons, a small number of cell population expressed smooth muscle cell markers(SMA, smooth muscle actin). Major reprogramming barriers, such as H3K9me3, were also present in the donor nucleus. Western blots demonstrated that the expression of H3K9me3 in SKPs was remarkably lower than that in BEFs. These data suggested that SKPs may be easily reprogrammed in NT.2. BEFs and SKPs were used as donor cells for SCNT to investigate the development of cloned embryos in vitro. Our results revealed that the embryos cloned from SKPs yielded significantly higher cleavage rates than those from BEFs did. The embryos from SKPs showed significantly higher cleavage rate than those from BEFs did(79.50% vs. 68.25%,P<0.01).The percentage of the embryos that developed to the blastocyst stage was significantly higher in the SKP group than in the BEF group(41.25% vs. 21.00%, P<0.01).The percentage of the hatched embryos in the SKPs was remarkably higher than that of the counterpart(18.75% vs 7.5%, P < 0.05). Moreover, the total number of cells of the SKP-cloned embryos in the blastocyst stage was significantly higher than that of the fibroblast-cloned embryos(P<0.05). These results showed that embryos cloned from SKPs exhibited better qualities than those from BEFs did. This finding indicated that using SKPs can generate excellent blastocysts.In our study, bovine SKPs were isolated from the skin. After SCNT occurred, embryos cloned from multi-potent SKPs showed higher developmental competence than that from BEFs. Therefore, SKPs can be used to enhance the efficiency of bovine SCNT.