Isolation of epithelial stem cells from dermis by a three-dimensional culture system
Isolation of epithelial stem cells from dermis by a three-dimensional culture system
复制标题
DOI：
10.1002/jcb.20757
复制
发表时间：
2006-05-01
影响因子：
4
通讯作者：
Huh, NH
中科院分区：
文献类型：
--
作者：
Medina, RJ;Kataoka, K;Huh, NH
皮肤是一种具有代表性的自我更新组织，含有细胞。尽管已经进行了许多尝试来定义和分离皮肤来源的干细胞，但建立一个简单可靠的分离程序仍然是一个有待实现的目标。在这里，我们报告了基于干细胞可能以不依赖于锚定的方式生长的假设，使用一种简单的选择方法从小鼠胚胎皮肤中分离出具有干细胞特性的细胞。我们将从小鼠胚胎真皮制备的单细胞悬浮液接种到温度敏感凝胶中，并在单层培养中繁殖所产生的菌落。命名为真皮衍生上皮祖细胞-1 （DEEP）的细胞显示上皮形态，并在250天内迅速生长到200多倍的水平。当细胞保持融合时，它们自发形成球状体，并在球状体中持续生长。免疫染色结果显示，所有克隆均阳性表达细胞角蛋白-8、-18、-19和E-cadherin，阴性表达细胞角蛋白-1、-5、-6、-14、-20、vimentin、nestin和ckit。此外，它们表达上皮干细胞标记物，如p63、整合素PI和S100A6。TGF - β培养后，部分DEEP-1细胞表达α -平滑肌肌动蛋白。当这些细胞被移植到成年SCID小鼠的各种器官时，一部分接种的细胞群获得了神经、肝脏和肾脏细胞的特性。这些结果表明，我们分离的细胞是上皮干细胞来源的，我们的新方法可用于从皮肤组织中分离多能干细胞。
Skin is a representative self-renewing tissue containing stern cells. Although many attempts have been made to define and isolate skin-derived stem cells, establishment of a simple and reliable isolation procedure remains a goal to be achieved. Here, we report the isolation of cells having stem cell properties from mouse embryonic skin using a simple selection method based on an assumption that stem cells may grow in an anchorage-independent manner. We inoculated single cell suspensions prepared from mouse embryonic dermis into a temperature-sensitive gel and propagated the resulting colonies in a monolayer culture. The cells named dermis-derived epithelial progenitor-1 (DEEP) showed epithelial morphology and grew rapidly to a more than 200 population doubling level over a period of 250 days. When the cells were kept confluent, they spontaneously formed spheroids and continuously grew even in spheroids. Immunostaining revealed that all of the clones were positive for the expression of cytokeratin-8, -18, -19, and E-cadherin and negative for the expression of cytokeratin-1, -5, -6, -14, -20, vimentin, nestin, a ckit. Furthermore, they expressed epithelial stem cell markers such as p63, integrin PI, and S100A6. On exposure to TGF beta in culture, some of DEEP-1 cells expressed alpha-smooth muscle actin. When the cells were transplanted into various organs of adult SCID mice, a part of the inoculated cell population acquired neural, hepatic, and renal cell properties. These results indicate that the cells we isolated were of epithelial stem cell origin and that our new approach is useful for isolation of multipotent stem cells from skin tissues.