Gene expression profiles of E8.5, E9.5 and E11.5 kidney precursors

Understanding processes how the early stage kidney precursor gives rise to metanephric mesenchyme, which is a committed progenitor cells of adult kidney is important for the regeneration of kidney in vitro. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of global gene expression profiles of the developing kidney precursors. Those gene expression data provides insights into not only the stage specific marker genes but also the signals working in each population, which should be informative for the directed differentiation of pluripotent stem cells in vitro. Osr1-GFP knock-in mice were used to isolate kidney precursor cells from embryos at E8.5, E9.5 and E11.5. At E9.5 and E11.5 embryos, to identify the differences between nephron progenitors and surrounding mesenchyme, nephron progenitor populations were further enriched by gating Osr1-GFP positive Integrin alpha8 positive Pdgfr alpha negative population and compared with Osr1-GFP positive cells other than that gate. RNA was isolated from cells and the gene expression profiles were determined by microarrays.阐明早期肾前体如何分化为后肾间充质（metanephric mesenchyme）——后者是成体肾脏的定向祖细胞——的过程，对于体外肾脏再生研究具有重要意义。将荧光激活细胞分选术（fluorescent activated cell sorting, FACS）与微阵列分析相结合，可为发育中的肾前体构建全基因组基因表达谱提供一种高效且功能强大的研究手段。该基因表达数据集不仅可揭示各发育阶段的特异性标记基因，还能阐明不同细胞群中的信号调控机制，可为多能干细胞（pluripotent stem cells）的体外定向分化提供重要参考。本研究使用Osr1-GFP敲入小鼠，从胚胎发育第8.5天（E8.5）、第9.5天（E9.5）及第11.5天（E11.5）的胚胎中分离肾前体细胞。针对E9.5和E11.5天的胚胎，为区分肾单位祖细胞与周围间充质，研究人员通过分选Osr1-GFP阳性、整合素α8（Integrin alpha8）阳性、血小板衍生生长因子受体α（Pdgfr alpha）阴性的细胞群，进一步富集肾单位祖细胞，并将其与该分选门之外的Osr1-GFP阳性细胞进行对比。从收集的细胞中提取RNA，通过微阵列分析测定基因表达谱。