Maintenance of functional stem cells in isolated and cultured adult intestinal epithelium
Maintenance of functional stem cells in isolated and cultured adult intestinal epithelium
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DOI：
10.1006/excr.1999.4483
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发表时间：
1999-06-15
影响因子：
3.7
通讯作者：
Potten, CS
中科院分区：
文献类型：
--
作者：
Booth, C;O'Shea, JA;Potten, CS
我们之前描述了一种成人大肠上皮原代培养的方法，表明干细胞在分离和培养过程中都存活下来。然而，由于不存在此类细胞的标记物，因此很难确认干细胞的存活——只能使用功能特性来定义它们。不幸的是，其中许多（例如分化、隐窝再生）在培养中不会发生，可能是由于条件不理想所致。为了解决这个问题，在免疫功能低下的小鼠中皮下培养新鲜分离和培养的小肠隐窝和大肠隐窝。所有最初形成的囊肿内衬有简单的上皮，逐渐变成多细胞并形成包含许多有丝分裂和凋亡的内陷。通过杯状细胞粘蛋白产生测定，上皮分化也很明显。粘蛋白成熟也是正常肠道的典型现象。管腔经常充满粘蛋白和凋亡小体。有趣的是，在表现出明显的隐窝样形态的移植物中，增殖区域位于结构的底部，杯状细胞朝向管腔，即典型的隐窝样形态。因此，功能性成体干细胞似乎能够在分离和组织培养中存活，从而允许器官型再生，可能涉及同源盒基因表达。现在这可能允许直接的干细胞表征和实验操作，例如转染，并可能最终允许移植和治疗性基因疗法。 (C) 1999 年学术出版社。
We have previously described a method far the primary culture of adult large intestinal epithelium, suggesting that stem cells had survived both the isolation and the culture procedures. However, as no markers for such cells exist, cofirmation of stem cell survival is difficult- only the functional properties can be used to define them. Unfortunately, many of these (e,g., differentiation, crypt regeneration) do not occur in culture, probably due to suboptimal conditions. To address this problem both freshly isolated and cultured small and large intestinal crypts were grown subcutaneously in an immunocompromized mouse. All initially formed cysts lined by a simple epithelium which gradually became multicellular and formed invaginations containing many mitoses and apoptoses. Epithelial differentiation, as assayed by Goblet cell mucin production, was also apparent. Mucin maturation was also typical of the normal intestine. The lumen was frequently filled with mucin and apoptotic bodies. Interestingly, in grafts displaying pronounced crypt-like morphology the regions of proliferation were situated toward the base of the structure and the Goblet cells toward the lumen, i.e., a typical crypt-like morphology. Hence, functional adult stem cells appear to survive isolation and tissue culture, permitting organotypic regeneration, possibly involving homeobox gene expression. This may now allow direct stem cell characterization and experimental manipulation, such as transfection, and may ultimately permit transplantation and therapeutic gene therapy. (C) 1999 Academic Press.