DataSheet1_Modelling renal defects in Bardet-Biedl syndrome patients using human iPS cells.pdf

Bardet-Biedl syndrome (BBS) is a ciliopathy with pleiotropic effects on multiple tissues, including the kidney. Here we have compared renal differentiation of iPS cells from healthy and BBS donors. High content image analysis of WT1-expressing kidney progenitors showed that cell proliferation, differentiation and cell shape were similar in healthy, BBS1, BBS2, and BBS10 mutant lines. We then examined three patient lines with BBS10 mutations in a 3D kidney organoid system. The line with the most deleterious mutation, with low BBS10 expression, expressed kidney marker genes but failed to generate 3D organoids. The other two patient lines expressed near normal levels of BBS10 mRNA and generated multiple kidney lineages within organoids when examined at day 20 of organoid differentiation. However, on prolonged culture (day 27) the proximal tubule compartment degenerated. Introducing wild type BBS10 into the most severely affected patient line restored organoid formation, whereas CRISPR-mediated generation of a truncating BBS10 mutation in a healthy line resulted in failure to generate organoids. Our findings provide a basis for further mechanistic studies of the role of BBS10 in the kidney.

巴德-毕得综合征（Bardet-Biedl syndrome, BBS）是一类纤毛病，可对包括肾脏在内的多种组织产生多效性影响。本研究对健康供者与BBS患者供者的诱导多能干细胞（iPS cells）的肾脏分化潜能进行了对比分析。对表达肾母细胞瘤1（Wilms' Tumor 1, WT1）的肾脏祖细胞开展高内涵成像分析后发现，健康对照组、BBS1、BBS2及BBS10突变组的细胞增殖、分化状态与细胞形态均无显著差异。随后，我们在三维肾脏类器官系统中对3株携带BBS10突变的患者细胞系进行了检测。其中携带最严重突变且BBS10表达水平低下的细胞系，虽可表达肾脏标志物基因，但无法形成三维类器官。另外两株患者细胞系的BBS10 mRNA表达水平接近正常水平，在类器官分化至第20天时即可生成多种肾脏细胞谱系。然而当培养延长至第27天时，其近端小管区域出现了退行性病变。向受影响最严重的患者细胞系中转入野生型BBS10后，可恢复其类器官形成能力；而在健康细胞系中通过成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR）介导引入截短型BBS10突变，则会导致其无法形成类器官。本研究结果为进一步探究BBS10在肾脏中的作用机制提供了重要研究基础。