Mouse embryonic stem cell-derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo
Mouse embryonic stem cell-derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo
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DOI：
10.1681/asn.2006101078
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发表时间：
2007-06-01
影响因子：
13.6
通讯作者：
Wilson, Patricia
中科院分区：
文献类型：
--
作者：
Vigneau, Cecile;Polgar, Katalin;Wilson, Patricia
后肾是输尿管芽和芽基之间相互作用的结果而发育的中胚层器官。胚胎干（ES）细胞衍生的祖细胞的产生提供了再生治疗的潜力，但往往受到肿瘤形成的发展的限制。因为短尾畸形（T）表示中胚层特化，所以将具有敲入功能性T基因座以及ROSA 26基因座中的lacZ的绿色荧光蛋白（GFP）的小鼠ES细胞系（LacZ/T/GFP）用于细胞选择和谱系追踪。在缺乏白血病抑制因子的情况下，小鼠ES细胞产生可分化为中胚层的胚状体。优化培养条件（4天，10 ng/ml激活素-A）以产生最大数量的肾祖细胞群，所述肾祖细胞群通过表达肾标志物钙粘蛋白-11、WT-1、Pax-2和Wnt-4的特异性组合来鉴定。通过FACS选择进一步富集LacZ/T/GFP+细胞。将LacZ/T/GFP+细胞注射到器官培养中的胚胎肾外植体中5天后，β-半乳糖苷酶免疫组织化学显示掺入到生肾区的胚基细胞中。在单次注射到发育中的活新生小鼠肾脏后，共定位研究表明LacZ/T/GFP+细胞稳定地整合到近端小管中，具有正常的形态和正常的碱性磷酸酶和水通道蛋白-1的极化，持续7个月，没有畸胎瘤形成。它的结论是，确定分化的胚胎干细胞成胚状体与激活素-A和选择T表达提供了一种手段，以分离和纯化肾近端小管祖细胞的再生治疗中安全使用的潜力。
The metanephric kidney is a mesodermal organ that develops as a result of reciprocal interactions between the ureteric bud and the blastema. The generation of embryonic stem (ES) cell-derived progenitors offers potential for regenerative therapies but is often limited by development of tumor formation. Because brachyury (T) denotes mesoderm specification, a mouse ES cell line with green fluorescence protein (GFP) knocked into the functional T locus as well as lacZ in the ROSA26 locus (LacZ/T/GFP) was used in cell selection and lineage tracing. In the absence of leukemia inhibitory factor, mouse ES cells give rise to embryoid bodies that can differentiate into mesoderm. Culture conditions were optimized (4 d, 10 ng/ml Activin-A) to generate maximal numbers of renal progenitor populations identified by expression of the specific combination of renal markers cadherin-11, WT-1, Pax-2, and Wnt-4. LacZ/T/GFP+ cells were further enriched by FACS selection. Five days after injection of LacZ/T/GFP+ cells into embryonic kidney explants in organ culture, beta-galactosidase immunohistochemistry showed incorporation into blastemal cells of the nephrogenic zone. After a single injection into developing live newborn mouse kidneys, co-localization studies showed that the LacZ/T/GFP+ cells were stably integrated into proximal tubules with normal morphology and normal polarization of alkaline phosphatase and aquaporin-1 for 7 mo, without teratoma formation. It is concluded that defined differentiation of ES cells into embryoid bodies with Activin-A and selection for T expression provides a means to isolate and purify renal proximal tubular progenitor cells with the potential for safe use in regenerative therapies.