Quantitation of human mammary epithelial stem cells with in vivo regenerative properties using a subrenal capsule xenotransplantation assay
Quantitation of human mammary epithelial stem cells with in vivo regenerative properties using a subrenal capsule xenotransplantation assay
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DOI：
10.1038/nprot.2010.148
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发表时间：
2010-12-01
期刊：
影响因子：
14.8
通讯作者：
Eaves, Connie J.
中科院分区：
文献类型：
--
作者：
Eirew, Peter;Stingl, John;Eaves, Connie J.
使用功能终点识别和计数正常组织中原始的、通常是罕见的未分化细胞的方法是获得对调节正常组织干细胞周转和分化的机制的深入了解的有力工具。在本文中，我们描述了一种基于异种移植的方案，该方案允许在从分离的正常人类乳腺组织中获得的异种细胞群中特异性地检测和量化具有体内组织再生特性的乳腺干细胞。这种方法包括将含有测试细胞的胶原凝胶与支持性成纤维细胞一起植入高度免疫缺陷、激素补充的小鼠的肾被膜下，然后在4周后，寻找具有体外克隆形成活性的再生人类细胞。输入的人类乳腺干细胞的量化是使用标准的限制稀释移植方法实现的。这种方法避免了修改小鼠乳房脂肪垫的需要，而且客观、快速(类似于5周)和经济。
Methods to identify and enumerate primitive, and typically rare, undifferentiated cells in normal tissue using functional endpoints are powerful tools for acquiring insights into the mechanisms that regulate normal tissue stem cell turnover and differentiation. In this paper, we describe a xenotransplantation- based protocol that allows mammary stem cells with in vivo tissue regenerative properties to be specifically detected and quantified among the heterogeneous cell populations obtained from dissociated normal human mammary tissue. This methodology involves implanting a collagen gel containing the test cells in combination with supportive fibroblasts under the kidney capsule of highly immune-deficient, hormone-supplemented mice and then, 4 weeks later, searching for regenerated human cells with in vitro clonogenic activity. Quantification of the input human mammary stem cells is achieved using standard limiting dilution transplant approaches. This approach circumvents the need to modify the mouse mammary fat pad, and is objective, rapid (similar to 5 weeks) and economical to perform.