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

Stem Cell Reports. 2016 Jan 12;6(1):18-25. doi: 10.1016/j.stemcr.2015.11.010. Epub 2015 Dec 24.


Multi-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques
  • Cell Differentiation / genetics
  • Cell Line
  • Cell Lineage / genetics
  • Cells, Cultured
  • Cilia*
  • Dyneins / metabolism
  • Endoderm / cytology
  • Endoderm / metabolism
  • Epithelial Cells / cytology*
  • Epithelial Cells / metabolism
  • Epithelial Cells / ultrastructure
  • GPI-Linked Proteins / metabolism
  • Gene Expression Regulation, Developmental
  • Humans
  • Intestines / cytology
  • Lung / cytology*
  • Metalloendopeptidases / metabolism
  • Microscopy, Electron
  • Microscopy, Fluorescence
  • Microtubules / metabolism
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Spheroids, Cellular / cytology*
  • Spheroids, Cellular / metabolism
  • Spheroids, Cellular / ultrastructure


  • GPI-Linked Proteins
  • carboxypeptidase M
  • Metalloendopeptidases
  • Dyneins