Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells

• CPM is a useful marker for generating human proximal airway epithelium
• Three-dimensional culture is useful for inducing human airway epithelium in vitro
• DAPT promotes the induction of multi-ciliated and pulmonary neuroendocrine cells
• Induced multi-ciliated airway cells have functionally motile cilia

SummaryMulti-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However, the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1+-ventralized anterior foregut endoderm cells (VAFECs), we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM+ VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore, the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT, a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a “9 + 2” microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine.

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