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.

Keywords: Human stem cells; Stem cells; iPS cells.

Figure 1. iAT2s in ALI culture express AT2 transcripts, tight junctions, and lamellar bodies.

(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.

Figure 2. iAT2 maturation in ALI culture is associated with decreased cell cycling.

(A) SPRING plot of iAT2s cultured in 3D, ALI, or 2D profiled by scRNA-Seq on day 10 after passage, colored by cell culture format. (B) Heatmap of top 10 differentially expressed genes per sample. (C) Violin plots of module scores for AT2 maturation genes, lamellar body–associated genes, and genes that are decreased with AT2 maturation (1-way ANOVA). (D) qPCR of SFTPA1, SFTPA2, SLPI, and SOX9 at 1, 3, 7, and 10 days after passage in iAT2s cultured at 3D or ALI (unpaired 2-tailed Student’s t test, n = 3). (E) SPRING plot of iAT2s cultured in 3D, ALI, and 2D profiled on day 10 after passage by scRNA-Seq, colored by inferred cell cycle phase; quantification of inferred cell cycle phase by cell culture format. (F) Flow cytometry plots of iAT2s exposed to a 24-hour pulse of EdU and collected on day 10 after passage (unpaired 2-tailed Student’s t test, n = 3). (G) Violin plots of AT2 maturation gene module score by cell cycle phase and cell culture format. Data are shown as mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Figure 3. iAT2s resemble primary AT2s in epithelial gene expression and differ in immune response state.

(A) Principal component analysis (PCA) of 10 samples (n = 3 per condition) showing global transcriptomic variance (%) of PC1 and PC2. Day 0, undifferentiated PSCs; day 15, day 15 NKX2.1⁺ progenitors; day 35 = day 35 tdTomato⁺ iAT2s; iAT2 3D, day 281 iAT2s, 10 days after passage in self-renewing 3D culture; iAT2 ALI, day 281 iAT2s, 10 days after passage to cell culture inserts; early HFL, weeks 16–17.5 (early canalicular) human fetal lung; late HFL, week 20–21 (late canalicular) human fetal lung; adult sorted AT2s, adult HTII-280–sorted AT2s; adult cultured AT2, adult HTII-280–sorted AT2s cultured in vitro; pediatric cultured AT2, pediatric HTII-280–sorted AT2s cultured in vitro. (B) Heatmap of row-normalized expression of key AT2 markers across iAT2 culture formats and primary lung samples. (C) Heatmap of Pearson correlation coefficients calculated between each sample based on normalized expression of human lung epithelial genes from an independent data set (25) and plotted as an average across samples for each group (see Supplemental Figure 4 for replicate values). (D) Heatmaps of row-normalized Z scores of top 100 differentially expressed genes between adult sorted AT2 versus iAT2 ALI (FDR < 0.05, ranked by log fold change) in the Hallmark gene sets Interferon Gamma Response, TNFA Signaling via NFkB, and Inflammatory Response.

Figure 4. iAT2s respond to combustible cigarette smoke and electronic cigarette vapor.

(A) SPRING plot of iAT2s cultured in 3D, 2D, or ALI and profiled by scRNA-Seq 12 hours after cigarette smoke exposure (Smoke) or air exposure (ALI), colored by condition. (B) Heatmap of top differentially expressed genes between cigarette smoke–exposed and air-exposed iAT2s in ALI culture. (C) Gene set enrichment analysis (GSEA, hypeR using Hallmark gene sets) of top upregulated genes in cigarette smoke–exposed iAT2s compared with air-exposed iAT2s (dotted line represents statistical significance threshold; FDR < 0.05). (D) qPCR of AKR1C3, CYP1A1, CXCL8, HMOX1, and NQO1 in iAT2s at 0, 2, 4, 6, 12, and 24 hours after exposure to combustible cigarette smoke or electronic cigarette vapor (unpaired 2-tailed Student’s t test, n = 3). (E) ELISA for IL-8 secreted by iAT2s into basolateral medium by 0, 2, 4, 6, 12, and 24 hours after exposure to combustible cigarette smoke or electronic cigarette vapor (unpaired 2-tailed Student’s t test, n = 3). Data are shown as mean ± SD. *P ≤ 0.05; **P ≤ 0.01.