Abstract:

Abstract:Objective To isolate the human keratinocyte stem cells(hKSCs) in vitro and evaluate  their biological characteristics. 
Methods The normal skin was taken by circum cision and the hKSCs were digested by enzyme and isolated by rapid adherence screening method. The morphology and ultrastructure of hKSCs were observed by phase contrast microscope and transmission electronic microscope,and the molecular markers were identified with immunocytochemistry and indirect immunofluorescence and the cell cycles were detected by flow cytometry.Results With phase contrast microscope,the rapidly adherent cells were  uniformed small, large nucleus  and epithelial-like. There were positive expressions of  cytokeratin 19,cytokeratin 15,p63 and CD29 by immunocy tochemistry,immunofluorescence  and flow cytometry, while there were negative expressions of vimentin and cytokeratin 10,which were the specific markers of fibroblasts and epidermal terminal differentiation respectively. The result of RT-PCR  was the same to the above. Most cells were in G0/G1 stage  by cell cycle analysis which suggested that hKSCs had slow cell cycling nature.  Conclusion hKSCs can be isolated and cultivated  in vitro successfully and they can be used as cell sources in the further experiments.

Key words: keratinocyte stem cells；isolation；biological characteristics