Air-liquid interface culture promotes maturation and allows environmental exposure of pluripotent stem cell–derived alveolar epithelium
Kristine M. Abo, … , Darrell N. Kotton, Andrew A. Wilson
Kristine M. Abo, … , Darrell N. Kotton, Andrew A. Wilson
Published March 22, 2022
Citation Information: JCI Insight. 2022;7(6):e155589. https://doi.org/10.1172/jci.insight.155589.
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Resource and Technical Advance Stem cells

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

Abstract

Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell–derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we 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. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.

Authors

Kristine M. Abo, Julio Sainz de Aja, Jonathan Lindstrom-Vautrin, Konstantinos-Dionysios Alysandratos, Alexsia Richards, Carolina Garcia-de-Alba, Jessie Huang, Olivia T. Hix, Rhiannon B. Werder, Esther Bullitt, Anne Hinds, Isaac Falconer, Carlos Villacorta-Martin, Rudolf Jaenisch, Carla F. Kim, Darrell N. Kotton, Andrew A. Wilson

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Figure 1

iAT2s in ALI culture express AT2 transcripts, tight junctions, and lamellar bodies.

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iAT2s in ALI culture express AT2 transcripts, tight junctions, and lamel...
(A) iAT2 differentiation and ALI culture schematic. iAT2s cultured as 3D spheres were dissociated and plated on cell culture inserts to generate ALI cultures. Apical medium was removed after 2 days in submerged culture. DE, definitive endoderm; AFE, anterior foregut endoderm. DS/SB = 2 μM dorsomorphin + 10 μM SB431542; CBRa = 3 μM CHIR99021 + 10 ng/mL BMP4 + 100 nM retinoic acid; CK = 3 μM CHIR99021 + 10 ng/mL KGF; DCI = 50 nM dexamethasone + 0.1 mM cAMP + 0.1 mM IBMX; and Y = 10 μM Rho-associated kinase inhibitor (Y-27632). (B) Bright-field and fluorescence imaging of iAT2s targeted with a tdTomato-encoding cassette to the endogenous SFTPC locus cultured in 3D and at ALI for 10 days. Scale bars: 1 mm. (C) Immunofluorescence images of pro-SFTPC (green) and SFTPC-tdTomato (red) in iAT2s cultured at ALI for 10 days. Scale bar: 25 µm. (D) Representative flow cytometry plots of NKX2.1 expression in iAT2 ALI cultures and quantification of NKX2.1 and SFTPC-tdTomato expression (n = 3; additional flow cytometry plots in Supplemental Figure 1). (E) Violin plot of AT2 differentiation module (CLDN18, LAMP3, NAPSA, SFTPB, SFTPC, SFTPD, and SLC34A2) score in iAT2s cultured in 3D, ALI, or 2D submerged conditions on day 10 after passage by scRNA-Seq (1-way ANOVA). (F) Transepithelial electrical resistance of iAT2 ALI or 2D submerged cultures 0–8 days after passage (unpaired 2-tailed Student’s t test, n = 3 per condition). (G) Electron micrograph of iAT2 ALI culture showing a tight junction (TJ) and lamellar bodies (LB). Scale bars : 500 nm. Data are shown as mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001.