mRNA sequencing of wildtype versus CEP83 knockout hiPSCs at diffirent time points of differentiation to kidney organoids [scRNA-seq]. 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蛋白的组成成分，对于初级纤毛的组装必不可少。既往研究表明，初级纤毛参与正常发育与组织稳态维持，其中包括肾脏发育。肾脏由人类诱导多能干细胞（human induced pluripotent stem cell，hiPSCs）发育而来，其发育过程呈阶梯式：首先在中间中胚层（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。本研究采用CRISPR-Cas9基因编辑技术，在hiPSCs中敲除CEP83，并将敲除后的hiPSCs与野生型hiPSCs均诱导分化为中间中胚层，随后进一步分化为肾脏上皮细胞，以此分析CEP83在人类肾脏上皮细胞分化过程中的作用。本研究通过形态学分析、基因表达检测及免疫染色技术，对中间中胚层与肾单位分化过程进行分析。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了侧板中胚层（lateral plate mesoderm，LPM）的调控转录因子，包括OSR1、FOXF1、FOXF2、HAND2及FEDRR，同时下调了特定肾单位祖细胞的标记基因，如PAX8、HOXB7与EYA1。进一步实验表明，野生型hiPSCs可成功分化为肾脏类器官，该类器官会上调关键肾单位标记基因，包括NPHS1、CUBN及GATA3。与之相反，CEP83敲除的hiPSCs无法分化为肾单位上皮细胞，且不表达肾单位标记基因。在基于hiPSCs构建的人类肾脏上皮细胞分化模型中，本研究数据为CEP83在肾单位祖细胞分化中的关键作用提供了新见解，或有助于进一步阐明CEP83相关肾脏发育缺陷的发病机制。实验整体设计：本数据集包含将hiPSCs向肾脏类器官分化第7天（中间中胚层阶段）的野生型细胞与CEP83敲除细胞的单细胞测序数据。