Wnt/β-catenin signaling is critical for regenerative potential of distal lung epithelial progenitor cells in homeostasis and emphysema

Abstract

Wnt/β-catenin signaling regulates progenitor cell fate decisions during lung development and in various adult tissues. Ectopic activation of Wnt/β-catenin signaling promotes tissue repair in emphysema, a devastating lung disease with progressive loss of parenchymal lung tissue. The identity of Wnt/β-catenin responsive progenitor cells and the potential impact of Wnt/β-catenin signaling on adult distal lung epithelial progenitor cell function in emphysema are poorly understood. Here, we used TCF/Lef:H2B/GFP reporter mice to investigate the role of Wnt/β-catenin signaling in lung organoid formation. We identified an organoid-forming adult distal lung epithelial progenitor cell population characterized by a low Wnt/β-catenin activity, which was enriched in club and alveolar epithelial type (AT)II cells. Endogenous Wnt/β-catenin activity was required for the initiation of multiple subtypes of distal lung organoids derived from the Wnt^low epithelial progenitors. Further ectopic Wnt/β-catenin activation specifically led to an increase in alveolar organoid number; however, the subsequent proliferation of alveolar epithelial cells in the organoids did not require constitutive Wnt/β-catenin signaling. Distal lung epithelial progenitor cells derived from the mouse model of elastase-induced emphysema exhibited reduced organoid forming capacity. This was rescued by Wnt/β-catenin signal activation, which largely increased the number of alveolar organoids. Together, our study reveals a novel mechanism of lung epithelial progenitor cell activation in homeostasis and emphysema.

Keywords: Wnt/β-catenin; chronic lung disease; emphysema; lung epithelial progenitor; organoid; regeneration.

Figure 1

The activity of Wnt/β-catenin signaling defines distal lung epithelial populations with distinct transcriptome. A, Experimental design and gating strategy for FACS sorting EpCAM⁺Wnt^neg/low/high fractions from TCF/Lef:H2B-GFP (TCF:GFP) mouse lungs. The proportion of the total EpCAM⁺ population represented by each fraction is shown. Mean ± SEM (N = 14). B, Expression of EGFP mRNA in the freshly sorted EpCAM⁺Wnt^high, Wnt^low, and Wnt^neg cells from the TCF:GFP mouse lung. Mean ± SEM (n = 6). Statistics were performed on ΔCT values (ΔCT = CT^HPRT - CT^target). *P < .05, **P < .01, ***P < .001, Student's t test. C, Heatmap of the expression of 19 112 mouse genes showing distinct transcriptome of the sorted Wnt^neg/low/high cells. D, Principle component analysis using the top 500 differentially expressed genes in the Wnt^{neg, low and high} cells (N = 6). EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting cells (Figure 1B) and thus refer to these cells as Wnt^neg/low/high cells from here on.

Figure 2

Airway and alveolar epithelial progenitors are enriched in the Wnt^neg and Wnt^low populations. A, Expression of lung epithelial cell markers in the Wnt^neg/low/high cells. Data presented is mean ± SEM of the expression Log2 value. N = 6. *P < .05 compared to other two groups. Student's t test. B, IF for CC10 (white), SFTPC (red, top panels), and ACT (red, bottom panels) on cytospins of FACS sorted Wnt^neg/low/high cells from adult TCF:GFP mouse lung. Scale bar = 50 μm. Nuclei stained with DAPI (blue). C, Quantification of CC10⁺, SFTPC⁺, and ACT⁺ cells in (B). Data shown is mean ± SEM. N = 4 individual animals. D,E, IF costaining of TCF:GFP lung sections for GFP (green) and lung cell markers (red). D, SFTPC (red, top) and ECAD (white, bottom). White arrowheads show GFP enriched in airway epithelium. E, An alveolar region, ECAD (white), SFTPC (red, top). Empty arrowheads show SFTPC⁺GFP^-cells adjacent to GFP⁺ (white arrows), probably nonepithelial cells. F, CC10 (red, top). White arrowheads: CC10⁺GFP^low cells, stars: CC10^-GFP^high cells. Scale bar = 50 μm. ACT, acetylated α-tubulin; DAPI, 4⁰,6-diamidino-2-phenylindole; ECAD, E-cadherin; FACS, fluorescence-activated cell sorting; IF, immunofluorescence

Figure 3

Organoid forming progenitor cells are enriched in the Wnt^low epithelial population. A, Schematic of organoid assay experimental setup. B, Organoid forming efficiency (Organoid count/ Number of seeded cells × 100, %) of sorted Wnt^neg/low/high fractions in the organoid assay. Data are shown as mean ± SEM (N = 20). Statistics were performed using the mean of triplicate or quadruplicate wells. **P < .01, one-way ANOVA with Bonferroni post-test. C, Representative IF images of alveolar (white arrows), bronchiolar (red arrow), and bronchioalveolar (pink arrow) organoids derived from the Wnt^low cells labeled by IF for SFTPC (white), ACT (red), GFP (green), and DAPI (blue). Scale bars = 50 μm. D, Quantification of each type of organoids formed by Wnt^low cells. Data are shown as mean ± SEM (N = 3). E,F, Pathways enriched in the Wnt^lowcells related to, E, epithelial progenitor and, F, stem cell functions. Plotted values are the -Log₁₀FDR values. Data shown on the right end of each bar: the numbers of significantly changed genes (both Wnt^low vs Wnt^neg and Wnt^low vs Wnt^high)/total number of genes in each pathway. ACT, acetylated α-tubulin; ANOVA, analysis of variance; DAPI, 4⁰,6-diamidino-2-phenylindole; FDR, false discovery rate; GFP, green fluorescent protein; IF, immunofluorescence

Figure 4

Wnt/β-catenin signaling is required for initial formation of alveolar organoids by Wnt^low cells but not for their subsequent proliferation. A,B, The effects of treatments with DMSO control, A, Wnt/ β-catenin inhibitor iCRT14 (10 μM) (N = 4) and, B, GSK3 inhibitor CHIR99021 (2 μM) (N = 4) added from day 0 on the organoid forming efficiency of Wnt^low cells. Data presented as mean ± SEM. ***P < .001, ****P < .0001, oneway ANOVA with Bonferroni post-test. C, Representative immunofluorescence images of day 14 organoids treated with DMSO, CHIR99021, and iCRT14, respectively. Scale bar = 1000 μm. D, Quantification of alveolar, bronchiolar, and bronchioalveolar organoids with DMSO/CHIR99021/iCRT14 treatments (N = 3). Mean ± SEM. *P < .05, **P < .01, Student's t test. E,F, Representative whole-mount immunofluorescence images showing GFP⁺ cells do not express proliferation marker Ki67 in organoid at, E, day 3 and alveolar organoids expressing SFTPC (white) at, F, day 5. Scale bars = 10 μm (E) and 20 μm (F). G, Effect of DMSO, iCRT14, or CHIR99021 on organoid diameter measured at day 14 (n > 120 organoids, N = 3). Mean ± SEM. *P < .05 compared to corresponding DMSO control, one-way ANOVA with Bonferroni post-test. ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; ns, nonsignificant

Figure 5

Wnt/β-catenin signaling attenuates the impaired organoid forming capacity of distal lung epithelial progenitors in emphysema. A, Pathway enrichment analysis of genes enriched in the Wnt^low cells but downregulated in COPD. Plotted values are -Log₁₀FDR. Data shown on the right ends of bars are numbers of genes that are enriched in Wnt^low cells but downregulated in COPD/ total number of genes in each pathway. B, H&E staining of lung tissue sections shows enlargement of airspace in the PPE treated lungs compared to those of saline treated mice after 21 days. Scale bar = 100 μm. C, Representative image of organoids at day 14 from PPE and saline treated lungs treated with DMSO and CHIR99021. Yellow arrows show organoids. Scale bar = 400 μm. D, Organoid forming efficiency of whole epithelial population from PPE and saline treated TCF:GFP mice at day 14 and effects of DMSO and CHIR99021 treatments added to the organoid culture at day 0. N = 6 individual animals per treatment. Data presented as mean ± SEM. ***P < .001, *P < .05, one-way ANOVA with Holm-Sidak post-test. E, Representative fluorescence images of small alveolar organoids (SFTPC⁺/ ACT^-, left, arrows) and large bronchiolar organoids (ACT⁺/SFTCP^-, right). Scale bar = 100 μm. Nuclei stained with DAPI (blue). F, Number of alveolar (SFTPC⁺/ACT^-) and bronchiolar (SFTPC^-/ACT⁺) organoids formed from 10 000 cells of each from PPE and saline treated TCF:GFP mice at day 14 and the effects of DMSO and CHIR99021 treatments added to the organoid culture from day 0. N = 4. Data presented as mean ± SEM. **P < .01, *P < .05, one-way ANOVA with Holm-Sidak post-test. ANOVA, analysis of variance; COPD, chronic obstructive pulmonary disease; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; FDR, false discovery rate; PPE, porcine pancreatic elastase