Directional Inducement to Osteoblasts of a Single Cell Cloning of Skin Stem Cells in Adult Cashmere Goat

Abstract: The skin pieces of cashmere goat were incubated in 0.02% protease at 4 ? overnight and the epidermis was separated from the dermis; the epidermal sheets were placed for 30 min in a sterile tube containing 0.25% trypsin at 37 ? and gently shaken to dissociate into single cell. The dissociated cells were allowed to adhere for 10 min at room temperature to culture dishes coated with 100 μg/ml collagen type IV. Cells that did not adhere in the allotted time were gently rinsed off each dish. Cultured the adhered cells in the cell culture incubator with 5% CO² at 37 ? for 4 days before replacing the medium with a fresh growth medium. Thereafter， change the medium every other day. At early confluence (80%-90% confluent) subculture the cells by detaching them by a brief treatment with the 0.25% trypsin solution. We isolated a single cell by diluting limitedly at second passage and cultured it in stem cell medium for a single cell cloning. Further identification by fluorescent-dye immunohistochemistry showed that they were positive for keratin15 and keratin19. Differentiation of epidermal stem cells toward the osteoblast lineage can be induced by supplementing medium with 50 μg/ml ascorbic acid， 10 mmol/L β-glycerophosphate and 1 μmol/L dexamethasone. The induced osteoblasts expressed positive by staining with 0.1% alizarin red and AKP. All results demonstrated that the isolated cells were skin stem cells which can be induced to osteoblasts.

CSTR: 32200.14.cjcb.2007.02.0018