Department of Pediatrics and Pediatric Stem Cell Research Institute, Sheba Medical Center, Israel.
Sackler School of Medicine, Tel Aviv University, Israel.
J Cell Mol Med. 2009 Aug;13(8B):1792-1808. doi: 10.1111/j.1582-4934.2008.00607.x.
During development, renal stem cells reside in the nephrogenic blastema. Wilms' tumour (WT), a common childhood malignancy, is suggested to arise from the nephrogenic blastema that undergoes partial differentiation and as such is an attractive model to study renal stem cells leading to cancer initiation and maintenance. Previously we have made use of blastema-enriched WT stem-like xenografts propagated in vivo to define a 'WT-stem' signature set, which includes cell surface markers convenient for cell isolation (frizzled homolog 2 [Drosophila] - FZD2, FZD7, G-protein coupled receptor 39, activin receptor type 2B, neural cell adhesion molecule - NCAM). We show by fluorescenceactivated cell sorting analysis of sphere-forming heterogeneous primary WT cultures that most of these markers and other stem cell surface antigens (haematopoietic, CD133, CD34, c-Kit; mesenchymal, CD105, CD90, CD44; cancer, CD133, MDR1; hESC, CD24 and putative renal, cadherin 11), are expressed in WT cell sub-populations in varying levels. Of all markers, NCAM, CD133 and FZD7 were constantly detected in low-to-moderate portions likely to contain the stem cell fraction. Sorting according to FZD7 resulted in extensive cell death, while sorted NCAM and CD133 cell fractions were subjected to clonogenicity assays and quantitative RT-PCR analysis, exclusively demonstrating the NCAM fraction as highly clonogenic, overexpressing the WT 'stemness' genes and topoisomerase2A (TOP2A), a bad prognostic marker for WT. Moreover, treatment of WT cells with the topoisomerase inhibitors, Etoposide and Irinotecan resulted in down-regulation of TOP2A along with NCAM and WT1. Thus, we suggest NCAM as a marker for the WT progenitor cell population. These findings provide novel insights into the cellular hierarchy of WT, having possible implications for future therapeutic options.
在发育过程中，肾祖细胞位于肾胚基中。Wilms 瘤（WT）是一种常见的儿童恶性肿瘤，据推测它起源于经历部分分化的肾胚基，因此是研究导致癌症起始和维持的肾祖细胞的理想模型。此前，我们利用体内增殖的富含胚基的 WT 类干细胞样异种移植物，定义了一个“WT-干细胞”特征集，其中包括用于细胞分离的细胞表面标志物（果蝇卷曲同源物 2 [FZD2]、FZD7、G 蛋白偶联受体 39、激活素受体 2B、神经细胞黏附分子-NCAM）。我们通过对球体形成的异质原发性 WT 培养物的荧光激活细胞分选分析表明，这些标记物中的大多数以及其他干细胞表面抗原（造血、CD133、CD34、c-Kit；间充质、CD105、CD90、CD44；癌症、CD133、MDR1；hESC、CD24 和假定的肾、钙黏蛋白 11），以不同水平在 WT 细胞亚群中表达。在所有标记物中，NCAM、CD133 和 FZD7 持续以低至中度水平检测，可能包含干细胞部分。根据 FZD7 进行分选导致广泛的细胞死亡，而分选的 NCAM 和 CD133 细胞部分进行克隆形成测定和定量 RT-PCR 分析，仅证明 NCAM 部分具有高度克隆形成能力，过度表达 WT“干性”基因和拓扑异构酶 2A（TOP2A），TOP2A 是 WT 的预后不良标志物。此外，用拓扑异构酶抑制剂依托泊苷和伊立替康处理 WT 细胞，导致 TOP2A 以及 NCAM 和 WT1 的下调。因此，我们认为 NCAM 是 WT 祖细胞群体的标志物。这些发现为 WT 的细胞层次结构提供了新的见解，可能对未来的治疗选择具有重要意义。
