Distinct Airway Epithelial Stem Cells Hide among Club Cells but Mobilize to Promote Alveolar Regeneration

Abstract

Lung injury activates specialized adult epithelial progenitors to regenerate the epithelium. Depending on the extent of injury, both remaining alveolar type II cells (AEC2s) and distal airway stem/progenitors mobilize to cover denuded alveoli and restore normal barriers. The major source of airway stem/progenitors other than basal-like cells remains uncertain. Here, we define a distinct subpopulation (∼5%) of club-like lineage-negative epithelial progenitors (LNEPs) marked by high H2-K1 expression critical for alveolar repair. Quiescent H2-K1^high cells account for virtually all in vitro regenerative activity of airway lineages. After bleomycin injury, H2-K1 cells expand and differentiate in vivo to alveolar lineages. However, injured H2-K1 cells eventually develop impaired self-renewal with features of senescence, limiting complete repair. Normal H2-K1^high cells transplanted into injured lungs differentiate into alveolar cells and rescue lung function. These findings indicate that small subpopulations of specialized stem/progenitors are required for effective lung regeneration and are a potential therapeutic adjunct after major lung injury.

Keywords: MHC(high) airway progenitors; alveolar injury and regeneration; bleomycin injury; dedifferentiation; lung epithelial stem cells; oxygenation; single cell transcriptomics; transplantation.

Figure 1:. Identification of non-BASC airway cell population with progenitor-like features.

(A) Cytospin analysis of EpCAM+/ITGB4+/CD200+ cell population enriched for epithelial progenitors identified ~45% cells stained with clara cell specific protein Scgb1a1, ~7% of cells were stained with Ac-Tubulin and Surfactant Protein C (SPC) each while ~40% of cells were unstained for mature lineage markers. ~25% of Scgb3a2+ cells were subpopulation of Scgb1a1+ cells. Scale bar = 25 μm. (B) Single cell transcriptomic sequencing of progenitor enriched cell population was performed on 10X Genomics platform. t-distributed stochastic neighbor embedding (tSNE) plot of single cells displays 8 distinct clusters. (C) Heatmap shows five top upregulated genes for each of the eight clusters identified through single cell mRNA sequencing of β4+/CD200+ cells. Four major types of cells were identified: Ciliated cells, p63+ cells, AEC2-like cells, and four clusters of club-like cells – all with high expression of Scgb1a1 and Cyp2f2 (club cell markers). (D) non-BASC airway epithelial cells (β4pos/SPC-CreERT2-neg) cells in 3-dimensional co-culture with freshly sorted mesenchyme (1:5 ratio) are the primary sources of largest colonies (n=3 replicates were analyzed). Scale bar = 150 μm. Area mean ± SD.**** p < 0.0001. Brown-Forsythe and Welch ANOVA tests followed by Holm-Sidak’s multiple comparisons test. (E) To identify minor population within the club cell like clusters with high progenitor activity, we performed supervised clustering using a Sox9-based progenitor gene signature of the bud tip cells from a developing mouse lung (Ostrin et al., 2018). (F) Progenitor genes used to re-cluster club like cells were enriched in clusters 0 and 3. See also Figures S1.

Figure 2:. Host defense-related signaling pathways highlight cells in progenitor cluster.

(A) Cluster 3 is negative or low for lineage markers of mature club (Scgb1a1, Scgb3a2) and alveolar epithelial type 2 cells (Sftpc). The cells in this cluster are marked by enhanced expression of surface markers Cd14, Cd74, H2-K1, and lncRNA AW112010. (B) Cells in cluster 3 are also enriched in genes associated with cell cycle in addition to cyclin inhibitors Cdkn1a and Cdkn1c. (C) Gene ontology analysis and pathway analysis identifies processes related with host-defense signaling pathways. Signaling pathways required for viral infection and replication are predicted to be activated in cells of cluster 3. Activation scores > 2 indicate a significant activation of a given pathway. (D) Consistent with activated host-defense pathway, cells in cluster 3 are highlighted by enhanced interferon regulated genes. (E) In situ staining for AW112010 lncRNA, which is expressed in H2-K1^high progenitor cluster, localizes the progenitor cells in the airway. AW112010 positive cells (red) with low or no detectable expression of club cell marker Scgb3a2 (white). The image is a composite image of multiple images taken at 20X and stitched together (See STAR Methods). See also Figures S2.

Figure 3:. H2-K1^high cells account for the in vitro regenerative activities of airway epithelium.

(A) High H2-K1 expression identifies a small fraction (~3%) of EpCAM+/β4+ cells via flow cytometry. Flow plot representative of >10 independent experiments. (B) β4+/H2-K1^high cells have the highest colony forming activity in vitro in mesenchyme free 3-dimensional culture with lung progenitor media (LPM). Lung progenitor media has key growth factors typically secreted from mesenchyme (Figure S3). Each data point represents a biological replicate. Data are presented as mean ± SD. **** p < 0.0001 (One-way ANOVA followed by Tukey’s multiple comparisons test). (C) β4+/H2-K1^high cells maintain their colony forming efficiency for at least three passages in culture. Each data point represents a biological replicate. (D) At the end of first passage, β4+/H2-K1^high colonies can differentiate towards either alveolar fate (SPC+) or airway fate (Krt5+ basal and Scgb3a2+ club cells). (E) Scgb1a1-CreERT lineage labeled at least three major populations in the lung epithelium as determined by cytospin analysis. Various cell types labeled by Scgb1a1-CreERT included β4+/H2-K1^high cells, which represented ~6% of total lineage labeled airway cells by Scgb1a1-CreERT. (F, G) Scgb1a1-CreERT lineage labeled H2-K1^high cells also account for all colony forming cells in (F) mesenchyme-free or (G) mesenchyme-dependent 3D culture conditions. β4+/H1-K1^high cells represent the cells with progenitor activity ascribed to lineage labeled club cells. See also Figure S3.

Figure 4:. β4+/H2-K1^high cells expand after injury and acquire senescence-associated markers during fibrotic phase of bleomycin injury.

(A) Fraction of β4+/H2-K1^high cells increases preferentially immediately after injury as determined by flow cytometry at 3 and 6 days after bleomycin injury. These expanded H2-K1^high cells gradually lose their ability to form large colonies in vitro at days 3, 6, and 9 post bleomycin injury. (data representative of n=2 independent experiments from n=3 mice each). (B, C) Single cell RNA-seq was performed on EpCAM+β4+/CD200+ cell population 9 days after injury followed by supervised clustering using the Sox9-based gene signature of progenitor cells (Figure 1D), which identified H2-K1^high cell cluster (labeled as Progenitors). Cells in this cluster expressed low levels of mature lineage markers such as Surfactant Protein C and secretoglobins (Scgb1a1 and Scgb3a2), as expected. These cells also express senescence associated markers such as Cdkn1a, Cdkn2a, Hif1a, and Trp53. (D) Injured β4+/H2-K1^high cells (d9 post injury) have increased expression of alveolar genes and decreased expression of airway genes when compared with uninjured β4+/H2-K1^high progenitor cells, suggesting acquisition of alveolar fate. (E) Pathway analysis of injured β4+/H2-K1^high cells in comparison with uninjured progenitor cells identify upregulation of signaling pathways associated with cellular movement while mature club cells from injured lungs remain unresponsive to the injury when compared to uninjured club cells. See also Figure S4.

Figure 5:. β4+/H2-K1^high cells can differentiate into alveolar cells in vivo.

(A) To understand lineage relationship between H2-K1^high progenitors from uninjured lungs and alveolar, club, and expanded H2-K1^high cells post injury, all uninjured club like cells (including H2-K1^high progenitors) were merged with bleomycin injured β4+/CD200+ cells followed by supervised clustering based on progenitor genes as used in Figure 1D. Four cell types are identified: H2-K1^high cells, Foxj1+ ciliated cells, Scgb1a1+ club cells, and Sftpc+ AEC2s (See Figure S5). (B) UMAP embedded merged objects shows either d0 (uninjured; green) or d9 bleomycin injured cells (red). Uninjured H2-K1^high (d0) progenitors are highlighted by blue circle. (C) RNA velocity was calculated to predict lineage differentiation of the merged objects and plotted onto the UMAP embedding. The RNA velocity shows three distinct lineages emanating from H2-K1^high cells: H2-K1^high cells to Sftpc+ AEC2s, H2-K1^high cells to Foxj1+ ciliated cells, and H2-K1^high cells into further H2-K1+ cells, some of which express AEC1 markers (Ager, Pdpn; Figure S5). (D) Sub-setting AEC1s, AEC2s, and Progenitors followed by re-calculation of RNA velocity identified a trajectory from uninjured progenitors to AEC1s and AEC2s. Uninjured H2-K1^high (d0) progenitors are highlighted by blue circle. (E) 75,000 freshly sorted Sox2-lineage labeled airway cells containing ~4% (or ~3000) H2-K1^high cells or 75,000 freshly sorted Sox2-lineage labeled cells depleted of H2-K1^high cells were transplanted 10 days after mice were injured with bleomycin. Scale bar = 1mm. Dotted line outlines an individual lobe. Sox2-lineage label excludes BASCs and includes H2-K1^high progenitors, P63+ LNEPs, other rare airway progenitors, and mature club cells. Whereas Sox2+/H2-K1^low includes all of those progenitors except the H2-K1^high progenitors. Total fluorescent intensity corresponding to transplanted cells was captured and quantified. 2-3 lobes/mouse were imaged from n=4 mice in each condition. Each data point represents quantification of fluorescent intensity from an individual lobe. Mann-Whitney test was used to determine significance. p-value < 0.05 is considered significant. Data are presented as mean ± SD. (F) A representative section of a mouse lobe transplanted with either Sox2-labeled or Sox2-labeled/H2-K1^neg cells is visualized showing limited engraftment of Sox2-labeled/H2-K1^neg cells. Image represents a composite of image of multiple images captured at 10X and stitched together (see STAR Methods). Scale bar = 1mm. (G) Sox2-labeled cells, which include H2-K1^high progenitors, have engrafted in injured lung and express pro-Surfactant Protein C (pro-SPC), indicative of their differentiation towards AEC2 fate. Image represents a composite of image of multiple images captured at 20X and stitched together (see STAR Methods). Scale bar = 200 μm. See also Figure S5 and S6.

Figure 6:. Expanded progenitor cells differentiate in vivo and aid in functional recovery of injury mice.

(A) Schematic experimental design. Freshly sorted progenitor cells from unlabeled SPC-CreERT2 mice were cultured in mesenchyme-free 3D conditions with LPM for 10 days. 100μM 4-Hydroxytamoxifen (4-OHT) was added in culture for the last 48 hours to label SPC expressing cells, which accounted for ~38% of all cells (see Figure 3D). Wild-type mice were injured with bleomycin on D0. Either 250,000 cells or saline were transplanted in injured mice at 10 days post injury and lungs were harvested on 24 days after initial injury. Oxygen saturation was measured at indicated time-points. (B) A representative section of a mouse lobe transplanted with either SPC-expressing (GFP+) or SPC-negative (tdTomato+) cells shows engraftment of tdTomato+ cells accounts for >90% of total engraftment areas. Boxed region is enlarged in C. Image represents a composite of image of multiple images captured at 10X and stitched together (see STAR Methods). (C) tdTomato+ cells, upon engraftment, differentiate into pro-SPC+ AEC2s and RAGE1+ AEC1s (same engrafted region from sequential section is shown). Image represents a composite of image of multiple images captured at 10X and stitched together (see STAR Methods). (D) Oxygen measure at day 0 (day of the injury) and starting day 10 post injury (day of the transplants) indicate improved oxygenation in mice that received transplant (n=4 for transplant and n=5 for non-transplant mice). Each data point represents oxygen saturation level of one mouse at the indicated time point. Mann-Whitney test was used to determine significance. * p < 0.05, **p < 0.01. Data are presented as mean ± SD.

Figure 7:. Injury-specific mobilization of distal airway stem/progenitor cells.

Distinct airway progenitors activate and expand depending on the type of injury. Newly identified H2-K1^high progenitors are transcriptionally highly similar to mature club cells but have unique regenerative characteristics that are identified through single cell mRNA-seq. These cells are preferentially targeted during viral injury, which leaves p63+ distal basal cells as the primary effectors of alveolar injury resolution. In bleomycin injury, p63^neg H2-K1^high progenitors, along with BASCs, are mobilized to promote regeneration of normal alveolar epithelium. See also Figure S7.