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  3. Protocol for the differentiation of human alveolar epithelial type I cells from pluripotent stem cell-derived type II-like cells

Protocol for the differentiation of human alveolar epithelial type I cells from pluripotent stem cell-derived type II-like cells

  • STAR Protoc. 2025 Mar 21;6(1):103667. doi: 10.1016/j.xpro.2025.103667.
Claire L Burgess 1,  Lauren J Ayers 2,  Kasey Minakin 1,  Konstantinos-Dionysios Alysandratos 1,  Xaralabos Varelas 3,  Darrell N Kotton 4
Affiliations

Affiliations

  • 1 Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118, USA; The Pulmonary Center and Department of Medicine, Boston University Chobanian, Avedisian School of Medicine, Boston, MA 02118, USA.
  • 2 Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118, USA; The Pulmonary Center and Department of Medicine, Boston University Chobanian, Avedisian School of Medicine, Boston, MA 02118, USA. Electronic address: ljayers@bu.edu.
  • 3 Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118, USA; The Pulmonary Center and Department of Medicine, Boston University Chobanian, Avedisian School of Medicine, Boston, MA 02118, USA; Department of Biochemistry and Cell Biology, Boston University Chobanian, Avedisian School of Medicine, Boston, MA 02118, USA.
  • 4 Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118, USA; The Pulmonary Center and Department of Medicine, Boston University Chobanian, Avedisian School of Medicine, Boston, MA 02118, USA. Electronic address: dkotton@bu.edu.
PMID: 40048418 DOI: 10.1016/j.xpro.2025.103667
Abstract

Pulmonary alveolar epithelial type I (AT1) cells have a flattened morphology to permit the diffusion of oxygen into the capillaries and historically have been difficult to isolate or maintain in culture. Here, we present a protocol for generating human alveolar type I-like cells (induced pluripotent stem cell-derived AT1s [iAT1s]) from induced pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) in vitro. We describe steps to plate iAT1s in either 3D or air-liquid interface cultures and to analyze or isolate iAT1s via flow cytometry. For complete details on the use and execution of this protocol, please refer to Burgess et al.1.

Keywords

Cell culture; Cell differentiation; Stem cells.

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