mRNA sequencing of wildtype versus CEP83 knockout hiPSCs at diffirent time points of differentiation to kidney organoids [scRNA-seq]

Centrosomal protein 83 (CEP83) is a component of the distal appendages proteins of centrioles, which is necessary for the assembly of primary cilia. Previous studies have implicated primary cilia in normal development and tissue homeostasis, including kidney development. The kidney develops from human induced pluripotent stem cell (hiPSCs) in a stepwise process involving induction of specialized Nephron progenitors (NPs) in the intermediate mesoderm (IM), followed by their differentiation into kidney epithelia. Here, we used CRISPR-cas9 technology to knockout CEP83 in hiPSCs that were differentiated versus the wildtype hiPSCs into IM followed by kidney epithelial differentiation to analyze the role of CEP83 in human kidney epithelial differentiation. We used morphological analyses, gene expression studies and immunostaining to analyze IM and nephron differentiation. Bulk RNA and single cell sequencing showed that CEP83-/- IM cells abnormally upregulate regulatory transcription factors of lateral plate mesoderm (LPM) including OSR1, FOXF1, FOXF2, HAND2 and FEDRR., and downregulate marker genes in specific NPs ncluding; PAX8, HOXB7 and EYA1. Further, wildtype hiPSCs successfully differentiated into kidney organoids that upregulate key nephron markers, including NPHS1, CUBN and GATA3. In contrast, CEP83-/- hiPSCs failed to differentiate into nephron epithelia and didn't express nephron marker genes. In a human system modeling kidney epithelial differentiation from hiPSCs, our data provide a new insight to the essential role of CEP83 in NPs differentiation and may help to better understand the pathogenesis of CEP83-associated renal developmental defects. Overall design: The provided data comprise single cell sequencing data from wildtype and CEP83 knockout cells at day 7 (intermediate mesoderm stage) of differentiation of hiPSCs toward kidney organoids.

中心体蛋白83（Centrosomal protein 83, CEP83）是中心粒远端附属结构的组成成分，对初级纤毛的组装不可或缺。既往研究已证实，初级纤毛参与正常发育与组织稳态维持，其中涵盖肾脏发育过程。肾脏由人类诱导多能干细胞（human induced pluripotent stem cell, hiPSCs）逐步发育形成，该过程首先需要在中间中胚层（intermediate mesoderm, IM）中诱导生成特化的肾单位祖细胞（Nephron progenitors, NPs），随后这些祖细胞分化为肾脏上皮细胞。本研究借助CRISPR-Cas9技术，对hiPSCs开展CEP83基因敲除操作，并以野生型hiPSCs作为对照，将两类细胞均诱导分化为中间中胚层，后续进一步定向分化为肾脏上皮细胞，以此解析CEP83在人类肾脏上皮分化过程中的功能。我们通过形态学分析、基因表达检测及免疫染色，对中间中胚层与肾单位分化进程进行系统分析。批量RNA测序与单细胞测序结果显示，CEP83敲除（CEP83-/-）的中间中胚层细胞异常上调侧板中胚层（lateral plate mesoderm, LPM）的调控转录因子，包括OSR1、FOXF1、FOXF2、HAND2及FEDRR，同时下调特定肾单位祖细胞的标记基因，如PAX8、HOXB7与EYA1。此外，野生型hiPSCs可成功分化为肾脏类器官，该类器官会上调包括NPHS1、CUBN与GATA3在内的关键肾单位标记基因。与之形成鲜明对比的是，CEP83-/- hiPSCs无法分化为肾单位上皮细胞，且不表达任何肾单位标记基因。在基于hiPSCs构建的人类肾脏上皮分化模型中，本研究数据为CEP83在肾单位祖细胞分化中的核心作用提供了全新认知，或有助于进一步阐明CEP83相关肾脏发育缺陷的发病机制。整体实验设计：本数据集包含hiPSCs向肾脏类器官分化第7天（中间中胚层阶段）的野生型与CEP83敲除细胞的单细胞测序数据。