Key to differentiation into multiciliated cells derived from human iPSC

A research group led by Haruka Yamaki (a graduate student at the time of the research, now an assistant professor at the Graduate School of Medicine), Program-Specific Research Center Assistant Professor Satoshi Konishi, and Professor Shimpei Gotoh of the Department of Clinical Application at the Center for iPS Cell Research and Application (CiRA), Kyoto University, has announced the identification of a specific surface marker for "deuterosomal cells," precursor cells that arise during the process of differentiating from human iPS cells into multiciliated cells of the airway epithelium. The group also clarified the onset mechanism of certain subtypes of the intractable disease Primary Ciliary Dyskinesia (PCD). The findings are expected to lead to the development of diagnostic and therapeutic methods for PCD. The results were published in Stem Cell Reports on March 19.

Airway epithelial cells are composed of various cell types including multiciliated cells and expel inhaled pathogens and foreign substances together with mucus. Airway epithelial cells serve as a biological defense mechanism, and their abnormalities cause chronic respiratory infections including PCD. PCD is a hereditary disease caused by mutations in genes associated with cilia, affecting approximately one in 20,000 people, with an estimated 5,000 patients in Japan. No effective treatment exists. While numerous causative genes have been reported, most are involved in the structure and movement of cilia, with only limited research into differentiation.

Among PCD subtypes, RGMC, a subtype in which the number of cilia is reduced, is thought to be caused by mutations in genes involved in cilia differentiation. Mutations in the CCNO gene are considered the cause, and that gene is believed to be involved in the amplification of centrioles, the structures that serve as the origin of cilia, though the detailed mechanism had remained unclear.

In the present study, the research group focused on the deuterosome-mediated pathway, one of the amplification pathways, which produces large numbers of centrioles. The structure known as the centriole is amplified and migrates toward the apical surface of the cell, where they mature into basal bodies that serve as the foundation of cilia.

Deuterosomes are structures that function as scaffolds for centrioles. Because the precursor cells of multiciliated cells that possess these structures, deuterosomal cells, appear only transiently during development and regeneration, detailed analysis had previously been difficult. The research group therefore used an established technology for inducing airway epithelial cells from human iPS cells and performed single-cell transcriptomic analysis to identify deuterosomal cells.

They confirmed that these cells transiently peak early in differentiation and subsequently transition into mature multiciliated cells. The group also discovered that CD36 is a protein specifically expressed in these cells and used it as a marker to isolate them. The isolated cells were confirmed to show high expression of relevant genes and high capacity for differentiation into multiciliated cells.

The group further generated iPS cells derived from PCD patients carrying CCNO mutations, as well as iPS cells in which this mutation had been repaired. Then they induced both to differentiate into airway epithelial cells for comparative analysis.

The results showed that in disease-specific iPS cell-derived airway epithelial cells, CCNO protein expression was reduced and multiciliated cell formation was severely impaired. In contrast, these impairments were recovered in gene-corrected iPS cell-derived cells, and the number of centrioles also increased. From this it was clarified that CCNO mutations cause impaired amplification of centrioles in the airway, leading to impaired multiciliated cell formation. Single-cell transcriptomic analysis was performed on airway epithelial cells derived from both the disease-specific iPS cells and the corrected iPS cells, and gene expression was compared in detail.

The results showed that in the corrected iPS cells, differentiation proceeded normally through deuterosomal cells into multiciliated cells, whereas in the disease-specific iPS cell-derived airway epithelial cells, a differentiation pathway that bypassed deuterosomal cells emerged and an abnormal cell population increased. Going forward, the group plans to use the CD36-based isolation method to further advance understanding of the pathology of the disease.

Journal Information
Publication: Stem Cell Reports
Title: Deuterosomal cells are the responsible lineage for multiciliogenesis in human airway differentiation
DOI: 10.1016/j.stemcr.2026.102860