Partial Maintenance and Long-Term Expansion of Murine Skin Epithelial Stem Cells by Wnt-3a In Vitro

CD49f+CD34+ cells, a skin epithelial stem cell (EpSC)-rich population, were prepared from adult mouse skin and cultured in the presence of Wnt-3a without feeder cells. CD34 expression was retained in about 10% of the cells, which had proliferated about 1,000-fold by day 10, although completely lost by day 14. CD49f+CD34+ cells sorted on day 10 retained canonical Wnt-responsiveness, proliferated markedly in the presence of Wnt-3a, maintained undifferentiated epithelial cell marker expression, and promoted hair follicle development in vivo. Those were subjected to a second 10-day culture with Wnt-3a and sorted, and then the same procedures were repeated a total of 15 times. CD49f+CD34+ cells obtained from each of those cultures retained the same EpSC characteristics as the original cells. CD34+ and CD34− cells were found to produce Wnt-3a and Wnt/β-catenin inhibitors, respectively. CD34+ cells resided as small cellular clusters surrounded by a large amount of CD34− cells. Furthermore, we found that exogenous Wnt-3a delayed the conversion of CD34+ cells to CD34− cells and induced CD34− cells to suppress the production of Wnt/β-catenin inhibitors, likely leading to generation of a microenvironment favorable for maintaining EpSCs. Our results suggest the possibility of partial long-term maintenance of EpSCs in vitro by Wnt-3a