Air-liquid interface culture promotes maturation and allows environmental exposure of pluripotent stem cell–derived alveolar epithelium

March 22, 2022

Kristine M. Abo,1,2 Julio Sainz de Aja,3,4,5 Jonathan Lindstrom-Vautrin,1 Konstantinos-Dionysios Alysandratos,1,2 Alexsia Richards,6 Carolina Garcia-de-Alba,3,4,5 Jessie Huang,1,2 Olivia T. Hix,1,2 Rhiannon B. Werder,1,2 Esther Bullitt,7 Anne Hinds,2 Isaac Falconer,8 Carlos Villacorta-Martin,1 Rudolf Jaenisch,6,9 Carla F. Kim,3,4,5 Darrell N. Kotton,1,2 and Andrew A. Wilson1,2
1Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, Massachusetts, USA. 
2The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA.
3Stem Cell Program and Divisions of Hematology/Oncology and Pulmonary & Respiratory Diseases, Boston Children’s Hospital, Boston, Massachusetts, USA. 
4Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. 
5Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. 
6Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. 
7Department of Physiology & Biophysics, Boston University, Boston, Massachusetts, USA. 
8Boston University School of Medicine, Boston, Massachusetts, USA. 
9Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

This study comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. They find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. Then they leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.


Type 2 alveolar epithelial cells (AT2s) reside in the distal lung, where they function as facultative progenitor cells of the alveolus, synthesize and secrete surfactant, and respond to inhaled immune stimuli (1). Their central role in the biology of this lung compartment makes the ability to model AT2 homeostatic or aberrant function in vitro a desirable goal for lung researchers. In vitro models of human epithelia in air-liquid interface (ALI) culture have served as useful platforms that promote the differentiation and maturation of epithelial cells and permit in vitro modeling of infections and environmental exposures (2–10). Primary human AT2s can be cultured and passaged in vitro as 3-dimensional (3D) organoids (11, 12) but are sourced from explant or donor lungs and tend to lose expression of AT2-specific surfactants when cultured in 2D monolayers (13). Human induced pluripotent stem cells (iPSCs) can be directed to differentiate to AT2s (iAT2s) in 3D spheres, where they transcriptomically resemble primary human AT2s (14–16) and recapitulate disease-specific phenotypes of the human subject of origin (14, 17). We and others recently reported the successful adaptation of iAT2s to ALI culture and the application of that system for disease modeling (18–20). Here, we provide in-depth characterization of iAT2s in ALI culture in comparison with fetal and adult primary AT2s. We transcriptomically profile iAT2s and find that, when cultured at ALI, they upregulate key markers of AT2 maturation as they downregulate cell cycle–associated transcripts relative to those cultured in 3D spheres. Finally, we demonstrate that iAT2s cultured at ALI respond to clinically significant, injurious environmental stimuli including cigarette smoke and electronic cigarette vapor.

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