Modelling ciliopathy phenotypes in human tissues derived from pluripotent stem cells with genetically ablated cilia.

Cilia are antenna-like organelles associated with a spectrum of human disease states. The functions of cilia remain incompletely understood, particularly in humans where cellular models do not recapitulate complex disease. Here we show that human pluripotent stem cells lacking the kinesin-2 subunits KIF3A or KIF3B, generated by CRISPR gene editing, establish a general tool for the study of ciliary function in tissue morphogenesis. Both KIF3A−/− and KIF3B−/− hPSCs lack cilia, yet remain self-renewing and pluripotent, enabling efficient derivation of diverse somatic cells. Derived tissues and organoids express multi-lineage phenotypes modeling the ciliopathy spectrum, including neuronal and nephron differentiation defects, and polycystic kidney disease. Modular culture conditions and biochemical analysis reveal that human cilia mediate a critical switch in hedgehog signaling during organoid differentiation, and constitutively release extracellular vesicles containing signaling molecules associated with these phenotypes. KIF3A−/− and KIF3B−/− hPSCs thus establish a human genetic system linking complex ciliary phenotypes with endogenous mechanisms, for therapeutics discovery and regenerative applications.

纤毛是一种类似天线的细胞器，与一系列人类疾病状态相关。纤毛的功能仍未被完全理解，尤其是在人类中，细胞模型无法重现复杂的疾病。在此我们表明，通过CRISPR基因编辑产生的缺乏驱动蛋白 - 2亚基KIF3A或KIF3B的人类多能干细胞，为研究组织形态发生过程中的纤毛功能建立了一种通用工具。KIF3A - / - 和KIF3B - / - 人类多能干细胞都缺乏纤毛，但仍能自我更新且具有多能性，从而能够高效地分化出多种体细胞。分化出的组织和类器官表达模拟纤毛病谱的多谱系表型，包括神经元和肾单位分化缺陷以及多囊肾病。模块化培养条件和生化分析显示，人类纤毛在类器官分化过程中介导了刺猬信号通路的关键转换，并持续释放含有与这些表型相关的信号分子的细胞外囊泡。因此，KIF3A - / - 和KIF3B - / - 人类多能干细胞建立了一个将复杂的纤毛表型与内源性机制联系起来的人类遗传系统，用于治疗发现和再生应用。