A New Isolation Method of Human Limbal Progenitor Cells by Maintaining Close Association with Their Niche Cells
A New Isolation Method of Human Limbal Progenitor Cells by Maintaining Close Association with Their Niche Cells
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DOI：
10.1089/ten.tec.2010.0609
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发表时间：
2011-05-01
影响因子：
3
通讯作者：
Tseng, Scheffer C. G.
中科院分区：
文献类型：
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作者：
Chen, Szu-Yu;Hayashida, Yasutaka;Tseng, Scheffer C. G.
在人类角膜上皮中，干细胞的自我更新和命运决定在利姆布斯中称为沃格特栅栏的小生境微环境中受到高度调节。在此，我们发现用分散酶消化，其切割基底膜，并没有去除整个基底上皮祖细胞。相比之下，胶原酶消化分离的集群不仅包括整个上皮祖细胞，但也密切相关的间充质细胞，因为更好地保存一些基底膜基质。胶原酶分离更多的基底上皮祖细胞，这是p63 α +和小的尺寸（直径8 μ m），并产生显着更多的全克隆和部分克隆3 T3成纤维细胞饲养层比分散酶。此外，胶原酶分离出更多的小的泛细胞角蛋白-/p63 α-/波形蛋白+细胞，其直径小至5 μ m，并且不均匀地表达波形蛋白、Oct 4、Sox 2、Nanog、Rex 1、巢蛋白、N-钙粘蛋白、SSEA 4和CD 34。它们之间的密切联系的维护导致在无血清，低钙培养基中的克隆生长，而胰蛋白酶/EDTA破坏这种关联导致没有克隆生长，除非与3 T3成纤维细胞饲养层共培养。类似地，在上皮剥脱的羊膜上，维持这种结合导致一致和稳健的上皮生长，这也被胰蛋白酶/EDTA废除。胶原酶分离的集群产生的上皮生长的直径显着更大，其单细胞产生更多的全克隆3 T3成纤维细胞饲养层比分散酶分离片。这种新的分离方法可用于探索角膜缘上皮干细胞如何受其天然小生境细胞的调控。
In human corneal epithelium, self-renewal and fate decision of stem cells are highly regulated in a niche microenvironment called palisades of Vogt in the limbus. Herein, we discovered that digestion with dispase, which cleaves off the basement membrane, did not remove the entire basal epithelial progenitor cells. In contrast, digestion with collagenase isolated on cluster consisting of not only entire epithelial progenitor cells but also their closely associated mesenchymal cells because of better preservation of some basement membrane matrix. Collagenase isolated more basal epithelial progenitor cells, which were p63 alpha+ and small in the size (8 mu m in diameter), and generated significantly more holoclones and meroclones on 3T3 fibroblast feeder layers than dispase. Further, collagenase isolated more small pan-cytokeratin-/p63 alpha-/vimentin+ cells with the size as small as 5 mu m in diameter and heterogeneously expressing vimentin, Oct4, Sox2, Nanog, Rex1, Nestin, N-cadherin, SSEA4, and CD34. Maintenance of close association between them led to clonal growth in a serum-free, low-calcium medium, whereas disruption of such association by trypsin/EDTA resulted in no clonal growth unless cocultured with 3T3 fibroblast feeder layers. Similarly, on epithelially denuded amniotic membrane, maintenance of such association led to consistent and robust epithelial outgrowth, which was also abolished by trypsin/EDTA. Epithelial outgrowth generated by collagenase-isolated clusters was significantly larger in diameter and its single cells yielded more holoclones on 3T3 fibroblast feeder layers than that from dispase-isolated sheets. This new isolation method can be used for exploring how limbal epithelial stem cells are regulated by their native niche cells.