基于hPSCs构建类器官探索FOXI1在肾脏集合管闰细胞发育中的作用

肾集合管闰细胞的正常发育对维持机体酸碱平衡具有重要意义。研究已表明肾集合管闰细胞来源于输尿管芽，但其发育过程及机制尚不清楚。申请人前期对小鼠肾脏的单细胞转录测序结果进行分析，发现成熟集合管闰细胞和主细胞均来自于主细胞样前体细胞，FOXI1是潜在决定前体细胞发育为闰细胞的关键转录因子。目前没有理想的实验模型用于研究集合管细胞发育，申请人前期利用人多能干细胞（hPSCs）成功构建了具有输尿管芽/集合管结构和功能的类器官模型，发现集合管上皮前体细胞自我调控不能分化为闰细胞，FOXI1的表达可诱导主细胞样前体细胞分化为闰细胞，提示FOXI1在闰细胞发育中具有重要作用。本项目将采用hPSCs构建类器官模型，结合转录测序和构建可诱导FOXI1表达、双荧光报告基因及CRISPRi人多能干细胞系，阐明FOXI1在肾集合管闰细胞发育中的作用及机制。项目的完成有助于促进肾集合管上皮细胞相关疾病和再生的研究。

The normal development of renal collecting duct intercalated cell plays an important role in maintaining whole organism acid-base balance. Previous studies have shown that the renal collecting duct intercalated cells originate from ureteric buds, however, processes and mechanisms of the development are still unclear. By using single-cell transcriptomic analysis of mouse kidneys, we found that mature intercalated cells and principal cells of collecting ducts were derived from principal cell-like precursor cells, and FOXI1 was a key transcription factor potentially determining the differentiation of precursor cells into intercalated cells. There is no ideal model for studying the development of collecting duct cells. We early generated ureteric bud/collecting duct organoids from human pluripotent stem cells (hPSCs), which have similar structures and functions of ureteric buds/collecting ducts. We found that precursor cells in the organoids were unable to differentiate into mature intercalated cells spontaneously, and expression of FOXI1 promoted formation of intercalated cells from principal cell-like precursor cells, which indicated that FOXI1 might play an important role in the development of intercalated cell. In this project, hPSCs-derived organoid models, applications of transcriptional sequencing, and generation of inducible FOXI1，a double-reporter and CRISPRi hPSC lines will be used, aiming to explore the important role of FOXI1 and potential mechanisms involved in the development of intercalated cells. The completion of the proposed project will provide insights for studying renal collecting duct diseases and regeneration.