Basic Research Of The Induction Of Human Epidermal Cells To Dedifferentiate Into The Epidermal Stem Cells

Objectives:To investigate the effect of bFGF on cell-fate determination of human epidermal cells and its signaling related mechanism in vitro,meanwhile make a comparison of the difference between epithelial stem cells and those derived from dedifferentiation,and try to search for a possibility for the dedifferentiated stem cells to be used in the wound repair and regeneration of skin tissues.Methods:The expression of cytokeratin10(CK10),an established marker of the differentiated epidermal cells,integrinβ₁,cytokeratin19(CK19),cytokeratin14(CK14), and PCNA,which were expressed by epidermal stem cells and their subpopulations has been analysed in fetal skins during various stages of the development using immunohistochemical methods.Meanwhile,improved collagenâ…£-coated adhesion method was used to isolate and culture the epidermal stem cells(ESCs)after neutron-protease selectively digested the dermo-epidermal junctions.Morphological features and identification of these immature cells were studied by immunochemistry and confocal microscopy analysis.After treated with extracts from 14-day-old mouse fetuses at the concentration of 10%(w/v),phenotypic changes and the cell-fate determination of ESCs were detected by flow FACS analysis and RT-PCR assays. Furthermore,human foreskin epidermal sheets were prepared as previously described, following a removal of basal stem cells using repeated collagenâ…£adhesion methods. Then,the treated ultrathin epidermal sheet was transplanted into the fresh full-thickness skin wounds measuring about 1 cm in diameter on the back of athymic BALB/c nude mice.Grafted biopsies were removed at day 3,5 and 7 post-grafting for histological and immunohistochemical staining,and flow FACS analysis.Additionally,HEKa cell line was incubated with bFGF(100ng/ml)for 36 to 48hr,then MTT assay, immunochemistry and confocal microscopy analysis,flow FACS,electron microscopy, semiquantitative RT-PCR,western-blot and telomerase activity assays(TRAP)were used to detected the the phenotypic changes and cell-fate determination of epidermal cells after bFGF treatment,simultaneously,making a comparison of the difference between epithelial stem cells and those derived from dedifferentiation.HEK cells with no intervention treatment were used as a control.Results:Both integrinsβ₁ and K19 were coexpressed in the undifferentiated epidermis.A specific,developmentally downregulation ofβ₁ expression was observed in the basal layer at the period of follicular keratinization.Coinciding with the differentiation of the epidermis,differential expression of K14 was observed in the basal and spinous layers,while K10 was confined to the differentiated spinous and granular layers.At the period of interfollicular keratinization,the epidermal progenitor cells mainly localized in the basal layer of epidermis and outer sheath,bulge and hair matrix of follicles,which were positive for integrinβ₁,CK19,CK14 and PCNA.Secondly, immunocytochemical analysis proved that ESCs and their subunits were positive for a6 andβ₁ integrin,CK19 and 14,p63,Nestin and PCNA,yet negative for CK10 expression. Double staining immunofluorescence demonstrated that markers such asα₆ integrin and CD71 was co-expressed in ESCs,and there will be important regional differences in the distribution ofα₆ integrin and CD71 in ESCs and transit amplifying cells(TA cells). Meanwhile,two-color flow cytometric analysis ofα₆ integrin and CD71 consistently revealed that after treated with an extract from mouse fetuses,the percent ofα₆⁺ CD71⁺ and CD71⁺ fraction increased and reached to 68.43%and 4.51%of the total isolated cells respectively.However,the percent of a₆⁺ CD71 fraction reduced from 22.49%to 10.92%determined by flow-cytometry.Moreover,the expression levels ofβ₁ integrin and some putative biological markers in human epidermal cells were analyses by RT-PCR detection after fetus extract treatment.The relative gene expressions ofβ₁ integrin,CK19 and CK10 increased in ESCs,whereas the expression of CK14 in keratinocytes stem cells was observed to be significantly suppressed when compared to the appropriate controls.After the establishment of the dedifferentiation model of human epidermal cells in vivo,morphological analysis using immunohistochemical and immunofluorescent staining indicated that the expression of CK14 and 19,p63,and PCNA was localized in spinous and granular layers at the early grafted stage,while CK10 antigen was detectable in middle and outer layers.With the elongation of grafted period,a developmentally specific up-regulation ofβ₁ integrin and CK19 was detected in the spinous and granular layers,when CK14,p63,and PCNA appeared extensively in the inner and middle layers of grafted biopsies with down-regulation of CK10 in the middle and outer layers.It was intrigue that some stem cell formed islands could be also seen in the spinous and granular layers between the basal layer and the stratum corneum, which were strongly positive forβ₁ integrin and cytokeratin 19.MTT-assay proved that the optimum cultural condition to induce the dedifferentiation of epidermal cells into their progenitors was to culture HEKa cells for 36 to 48 hr with the addition of bFGF at a dosage of 100ng/ml.After treatment with bFGF for 48hr,clusters of round-shaped cells became detectable around differentiated epidermal cells,and expanded progressively thereafter.These cells were smaller in shape and mononucleated cells with large nuclear-cytoplasmic ratio,which led to not only clonogenicity but also ability to reconstitute an epidermal ridge like structure.Immunohistochemical staining revealed that the expression levels ofβ₁ integrin,CK19 and CK14 were up-regulated,while the expression of CK10 was significant down-regulated after bFGF treatment.For flow FACS analysis,essentially all cells were strong CK14-staining compared with controls (87.14 and 67.26%,respectively).Significantly,the fluorescence intensity of the CK19 population was upregulated from 15.74%to 74.77%,and which of the CK10 population decreased from 98.56%to 4.56%after bFGF treatment.Additionally,the expression levels ofβ₁ integrin and some putative biological markers in human epidermal stem cells were analyses by RT-PCR detection after bFGF treatment.The relative gene expressions ofβ₁ integrin,CK19 and CK14 increased in bFGF treatment group, whereas the expression of CK10 was observed to be significantly suppressed when compared to controls.Western-blot and TRAP also proved the dedifferentiated phenomenon of epidermal cells in our experimental system.When epithelial stem cells are compared with dedifferentiated stem cells treated with bFGF(dHEK cells),two points seem noteworthy.First,in both cases the cells remain multipotent and express markers of keratinocyte stem cells.Second,in both cases there seems to exist the regional differences by detection ofα₆ integrin and CD71 double labeling.However, although there seemed no significant changes were observed regarding the ultra and three-dimensional structure of ESCs and dedifferentiated derived stem cells following analysis with electron microscopy,double-immunofluorescent staining showed that the percents of TA cell subpopulation in dedifferentiated epidermal stem cells treated with bFGF is much higher than that in ESCs.Importantly,there exists a differentance of subcellular localization and hTERT activity between ESCs and dedifferentiated derived stem cells.Conclusion:bFGF can reverse the differentiated process of epidermal cells and induce them to produce immature,stem-like cells,which can proliferate and be used in the wound repair and regeneration of skin tissues,however,the mechanism involved in bFGF signaling on the induction of epidermal cells' dedifferentiation is unclear,which needs further research.