Isolation and cultivation of keratinocyte stem-like cells from human skin dermis

doi:10.3969/j.issn.2095-4344.2013.23.012

Abstract

Abstract:

BACKGROUND: Skin dermis is an essential stem cell source, except basal layer of epidermis, for forming epidermis structure during the process of skin wound healing.
OBJECTIVE: To explore a new method for isolating and culturing keratinocyte-stem like cells from human skin dermis.
METHODS: Definitive sizes of full skins from adult abdomen were digested with dispase overnight and the epidermal structure was discarded. The remaining dermis were digested with 0.1% type I collagenase overnight. Thereafter, the digested cells were collected by centrifugation, suspended in serum-free Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (1:1) containing 1% N₂, 2% B27, 20 μg/L epidermal growth factor, 40 μg/L basic fibroblast growth factor, and cultured at a low cell density. The isolated and cultivated cells were analyzed by reverse transcription-polymerase chain reaction and immunofluorescence analysis.
RESULTS AND CONCLUSION: The reverse transcription-polymerase chain reaction analysis showed that, the cells isolated from dermis contained hair follicle stem cells expressing Lgr5 and Lirg1. The immunofluorescence analysis showed that, the cells isolated from dermis expressed cytokeratin 8 and cytokeratin 14. After cultivated in serum-free media for 6 days, some cytokeratin 14 and integrin-α6 positive cell colonies appeared “paving stone” like morphology. The passage 1 cells expressed keratinocyte stem cell markers, such as cytokeratin 14, CD29 and P63. The cell proliferation rate was declined with passage times and almost all cells were terminally differentiated at passage 3 or 4. Experimental findings indicate that, keratinocyte stem-like cells expressing CD29, cytokeratin 14 and P63 can be successfully isolated from human skin dermis by directly cultured at a low density and in serum-free media.

Key words: stem cells, stem cell culture and differentiation, human skin, dermis, sweat gland, hair follicle, keratinocyte, cytokeratin 14, cytokeratin 8, CD29, gene, other grants-supported paper, stem cell photographs-containing paper

CLC Number:

R394.2

Chi Guang-fan, Li Mei-ying, Zhao Gui-fang, Deng Ji-Hong, Liu Jin-yu, Li Yu-lin . Isolation and cultivation of keratinocyte stem-like cells from human skin dermis[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(23): 4264-4271.

Figures/Tables 6

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