Isolation, Purification, Identification And Directional Inducement Of Stem Cells From Adult Cashmere Goat Skin

In this study, we investigated the characteristics and the avenues of isolation, purification, identification and directional inducement of stem cells from adult cashmere goat skin.The skin sample of cashmere goat was incubated in 0.02% protease at 4℃overnight, and the epidermis and hair follicles were separated; then they were placed in a sterile tube containing 0.25% trypsin at 37℃for 30 min and the dissociated cells were adhered with collagen type IV to culture dishes for 10 min. 10min later, the unattached cells were removed and the rapidly adherent epidermal cells were cultured in stem cell growth medium. The cells showed a large nuclear: cytoplasmic ratio and small size, and can formed different shapes of clones. The cell cycle profiles for different passages suggested most of the rapidly adherent epidermal cells arrested at G1/G0 phase and they had a higher proliferative potential. Moreover, the isolated cells expressed k15, k19, p63, CD34 andβ1-Integrin. These results indicated that there were epidermal stem cells in the population of the rapidly adherent epidermal cells.Then, a single cell was isolated using 96-hole culture plate by diluting limitedly at the second passage of the rapidly adherent epidermal cells, and cultured for a single cell cloning and expanded in stem cell medium. The cells had the stem cell characteristics of immaturity, round, small size, few organelles, a high nuclear:cytoplasmic ratio, and they could form holoclone. The cells were slow cycle and most stayed at G1/G0 phase. The CFE of the cells was higher, and they possessed high proliferative capacity and strong clonogenicity in vitro. To evaluate the purified cell populations, further identification by immunocytochemistry and fluorescence in situ hybridization were performed, the results indicated they were positive for Keratin15, Keratin19, p63, CD34 andβ1-integrin, but negative for Keratin10 and Involucrin. This implied that the purifed cells can be skin stem cells. In vitro, the skin stem cellsof cashmere goats can differentiate into the cells with characteristics of cardiomyocytes, nerve, osteoblast, which suggested that the purifed cells had a big plasticity.Furthermore, the stem cells in cashmere goat skin were located primarily by FISH.β1-integrin were expressed positively in the outer root sheath of hair follicle and the papillae in cashmere goat skin, but CD34 were positive in the basal layer of epidermis, the outer root sheath of hair follicle and the papillae.