Isolation, culture and phenotypic investigation of dedifferentiation-derived epidermal stem cells / 中华创伤杂志

ABSTRACT

Objective To isolate the dedifferentiation-derived epidermal stem cells (DDESCs)to further investigate their phenotypic characteristics. Methods The sheets of human foreskin were digested overnight after removal of adipose tissues and then the epidermis was separated from the dermis.The epidermis sheets which eliminated basal stem cells by repeated adhesion to type Ⅳ collagen and flushing were transplanted onto the full-thickness skin wounds on the back of BALB/c nude mice. After five days, the sheets were collected and digested into single cells, after which the percentages of positive cells of CK10, CK19 and β1 integrin were detected by flow cytometric analysis. DDESCs were isolated by rapid adhesion to type Ⅳ collagen. The expressions of CK19, β1 integrin, Oct4 and Nanog in the cells were examined using immunofluorescence and quantitative real-time polymerase chain reaction (RTPCR). Results The percentages of positive cells of CK19 and 31 integrin were increased (P ＜0.01 )and those of CK10 in the transplanted sheets decreased ( P ＜0.01 ) five days after transplantation. Isolation of DDESCs by repeated adhesion to type Ⅳ collagen showed 4.56% adhering cells in the transplantation group within 10 minutes. The in vitro phenotypic assays showed that the expressions of CK19, β1 integrin, Oct4 and Nanog in DDESCs were similar to those of original epidermal stem cells ( P ＞0.05 ) but remarkably higher than those in the control group ( P ＜ 0.01 ). Conclusion The phenotypic characteristics of DDESCs cultured in vitro are similar to those of epidermal stem cells, indicating a new approach for wound repair and regeneration.