Partial Maintenance and Long-Term Expansion of Murine Skin Epithelial Stem Cells by Wnt-3a In Vitro
Partial Maintenance and Long-Term Expansion of Murine Skin Epithelial Stem Cells by Wnt-3a In Vitro
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DOI：
10.1038/jid.2014.510
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发表时间：
2015-06-01
影响因子：
6.5
通讯作者：
Yoshikawa, Masahide
中科院分区：
文献类型：
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作者：
Ouji, Yukiteru;Ishizaka, Shigeaki;Yoshikawa, Masahide
从成年小鼠皮肤制备CD 49 f（+）CD 34（+）细胞，一种富含皮肤上皮干细胞（EpSC）的细胞群，并在Wnt-3a存在下培养而不培养饲养细胞。CD 34表达保留在约10%的细胞中，其在第10天增殖约1，000倍，尽管在第14天完全丧失。在第10天分选的CD 49 f（+）CD 34（+）细胞保持典型的Wnt-反应性，在Wnt-3a存在下显著增殖，维持未分化上皮细胞标记物表达，并促进体内毛囊发育。用Wnt-3a进行第二次10天培养并分选，然后重复相同的程序总共15次。从每种培养物中获得的CD 49 f（+）CD 34（+）细胞保留了与原始细胞相同的EpSC特征。发现CD 34（+）和CD 34（-）细胞分别产生Wnt-3a和Wnt/β-连环蛋白抑制剂。CD 34（+）细胞以小细胞团的形式存在，周围有大量的CD 34（-）细胞。此外，我们发现外源性Wnt-3a延迟了CD 34（+）细胞向CD 34（-）细胞的转化，并诱导CD 34（-）细胞抑制Wnt/β-连环蛋白抑制剂的产生，可能导致产生有利于维持EpSCs的微环境。我们的研究结果表明，部分长期维持的体外EpSCs的Wnt-3a的可能性。
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/beta-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/beta-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.