Isolation And Characterization Of SSEA3~+Stem Cells Derived From Goat Skin

Adult stem cells (ASCs) are multipotent stem cells which are resided in fetus and adulttissues and have the ability of self-renewal and multiple differentiation potential. These cellshave a promising future in the field of life science and regenerative medicine. Previousstudies have demonstrated that novel stem cells expressing the pluripotency marker ofanti-stage-specific embryonic antigen3(SSEA3) reside among human dermal fibroblasts, andthese SSEA3~+cells are stressful and named Multilineage-differentiating stress-enduring(Muse) cell. These cells are able to self-renew, express pluripotency markers, and differentiateinto endodermal, ectodermal, and mesodermal cells both in vitro and in vivo. Furthermore,SSEA3~+cells can enhance induced pluripotent stem cells (iPSCs) generation efficiency, withMuse-iPSCs demonstrating a formation efficiency of0.03%,30-fold more efficiently thanthat for fibroblasts (0.001%), and whereas iPSCs could not be derived from SSEA3-cells.These findings provide a new perspective for the research of cell reprogramming. However,SSEA3~+cells have only been found in humans.In the present study, SSEA3~+cells were isolated from Saanen dairy goat skin fibroblastsby suspension culture and fluorescence-activated cell sorting (FACS). A suitable culturesystem in vitro was established for SSEA3~+cells. Furthmore, characterization of goat SSEA3~+cells were determined by using alkaline phosphatase staining, immunofluorescence staining,RT-PCR, flow cytometry and differentiation in vitro and in vivo. These results have providedthe basis and materials for the research of mammal adult stem cells and transgenic animalproduction. Results are as follows:1. Approximately3-4%SSEA3~+cells from three different goat skin fibroblast cell lineswere isolated by suspension culture and fluorescence-activated cell sorting (FACS). Theseresults stated that a subpopulation of SSEA3~+cells were resident among goat skin fibroblasts.2. Stress induction assay showed that8-12h trypsin incubation was helpful to enrich theSSEA3~+cells, and about13%of fibroblasts with8-12h trypsin incubation positive forSSEA3by flow cytometry analysis. Furthermore, traditional method of trypsinization was notefficient for passage, whereas the recycling process of suspension culture and adherent culturewas more suitable for self-renew. These findings would provide basic foundation for isolationand culture of mammal adult stem cells. 3. Research results showed that goat SSEA3~+cells were positive for ALP staining andpluripotency markers Nanog, Oct4, Sox2, TRA-1-60and SSEA3, but were negative forSSEA1and SSEA4; furthermore, they did not express CD34, CD271, Snail, Slug exceptCD105(a marker of mesenchymal stem cell). And they are able to differentiate into all threegerm layers cells both in vitro and in vivo. All these demonstrated that goat SSEA3~+cells arespecial kind of pluripotent stem cell in skin.