Embryonic tissue extracts modulate cell-fate determination in human epidermal stem cells

AIM: To investigate the phenotypic changes and cell-fate determination of keratinocyte stem cells in vitro. METHODS: Improved collagen Ⅳ-coated adhesion method was used to isolate and culture the keratinocyte stem cells 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 keratinocyte stem cells (KSCs) were detected by flow FACS analysis and RT-PCR assays. RESULTS: Immunocytochemical analysis proved that KSCs and their subunits were positive for α₆ and β₁ integrin, keratin 19 and 14, p63, nestin and PCNA, yet negative for keratin 10 expressions. Double staining immunofluorescence demonstrated that markers such as α₆ integrin and CD71 was co-expressed in KSCs, and there were important regional differences in the distribution of α6 integrin and CD71 in KSCs and transit amplifying cells (TA cells). Meanwhile, two-color flow cytometric analysis of α₆ integrin and CD71 consistently revealed that after treated with an extract from mouse fetuses, the percent of α+6 CD71+ and CD71+ fraction increased and reached to 68.43% and 4.51% of the total isolated cells, respectively. However, the percent of α+6 CD71- fraction reduced from 22.49% to 10.92% determined by flow cytometry. Moreover, the expression levels of β₁ integrin and some putative biological markers in human epidermal cells were analyses by RT-PCR after fetus extract treatment. The relative gene expressions of β₁ integrin, K19 and K10 increased in KSCs, whereas the expression of K14 in keratinocyte stem cells was observed to be significantly suppressed when compared to the appropriate controls. CONCLUSION: The keratinocyte stem cells isolated by collagen Ⅳ-coated adhesion method have some characteristics of adult stem cells and perhaps the potentiality to differentiate and cross-differentiate. The regional differences in the distribution of α6 integrin and CD71 are one of the transition markers to discriminate the KSCs and TA cells. Furthermore, fetus extracts contribute to not only the proliferation and self-renewal of KSCs but also the dedifferentiation of TA cell subunits.