B28-11 Modeling of Primary Ciliary Dyskinesia With Defective Multiciliogenesis Using Patient-Derived iPS Cells

Abstract Rationale Primary ciliary dyskinesia (PCD) is a hereditary disorder caused by pathogenic variants in genes associated with motile cilia structure and biogenesis, leading to impaired mucociliary clearance, chronic airway infection, and bronchiectasis. The regulatory mechanisms governing multiciliogenesis and their disruption in PCD, particularly in the subtype characterized by reduced generation of multiple motile cilia (RGMC), remain poorly understood. Recent single-cell RNA sequencing (scRNA-seq) studies of human airway epithelium have identified deuterosomal cells (DCs) as transient precursors of multiciliated cells (MCCs). However, because DCs appear only transiently during development or epithelial regeneration and are rare in steady-state tissues, their molecular characteristics in humans remain largely unexplored. To address this, we used human induced pluripotent stem cell (iPSC)-derived airway epithelial cells to characterize human DCs and to investigate defective multiciliogenesis in PCD. Methods We established a strategy to isolate DCs from human iPSC-derived airway epithelial cells (iAECs) by using a cell surface antigen as a marker. To investigate the role of Cyclin O (CCNO), a gene specifically expressed in DCs, we generated iPSCs from a patient with PCD harboring pathogenic compound heterozygous CCNO variants and created isogenic gene-corrected control lines. Each iPSC line was differentiated into iAECs and analyzed to delineate phenotypic and transcriptional changes associated with CCNO deficiency. Results Isolated iPSC-derived DCs exhibited transcriptional profiles consistent with transient MCC precursors identified in primary human airway epithelium, enabling detailed molecular analysis of this short-lived population in vitro. Comparative scRNA-seq and immunofluorescence analyses of CCNO-deficient and gene-corrected iAECs showed reduced expression of cell cycle-related genes and proteins in CCNO-deficient DCs. Pseudotime analysis further revealed an abnormal differentiation trajectory in which cells bypassed the DC state and directly progressed toward immature multiciliated-like cells. Consequently, the proportion of mature MCCs with multiple motile cilia was markedly reduced, faithfully recapitulating the RGMC subtype of PCD in vitro. Conclusion We developed a human iPSC-based model of the RGMC subtype of PCD, which enabled identification and molecular characterization of DCs, revealing defective MCC differentiation in CCNO-related PCD. This study provides new insights into the mechanisms of human multiciliogenesis and contributes to understanding the pathogenesis of PCD. This abstract is funded by: JSPS KAKENHI (JP22H03077, JP23K24338, JP25K12809, and JP25K02660); AMED (JP19ek0109410, JP23bm1323001, and JP25bk0104190); the Fujiwara Memorial Foundation; the Naito Foundation; and the iPS Cell Research Fund for CiRA at Kyoto University.