
==== Front
Stem Cell Reports
Stem Cell Reports
Stem Cell Reports
2213-6711
Elsevier

S2213-6711(24)00116-4
10.1016/j.stemcr.2024.04.009
Article
Characterization of perivascular alveolar epithelial stem cells and their niche in lung homeostasis and cancer
Chen Qian 16
Hirai Hiroyuki 16
Chan Manwai 2
Zhang Jilei 1
Cho Minsu 2
Randell Scott H. 3
Kadur Lakshminarasimha Murthy Preetish 1
Rehman Jalees 245
Liu Yuru yuruliu@uic.edu
1257∗
1 Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL 60612, USA
2 Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, IL 60607, USA
3 Marsico Lung Institute, University of North Carolina School of Medicine, Chapel Hill, NC, USA
4 Department of Biochemistry and Molecular Genetics, University of Illinois College of Medicine, Chicago, IL 60607, USA
5 University of Illinois Cancer Center, Chicago, IL 60612, USA
∗ Corresponding author yuruliu@uic.edu
6 These authors contributed equally

7 Lead contact

16 5 2024
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18 4 2024
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© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Lung alveolar structure and function are maintained by subsets of alveolar type II stem cells (AT2s), but there is a need for characterization of these subsets and their associated niches. Here, we report a CD44high subpopulation of AT2s characterized by increased expression of genes that regulate immune signaling even during steady-state homeostasis. Disruption of one of these immune regulatory transcription factor STAT1 impaired the stem cell function of AT2s. CD44high cells were preferentially located near macro- blood vessels and a supportive niche constituted by LYVE1+ endothelial cells, adventitial fibroblasts, and accumulated hyaluronan. In this microenvironment, CD44high AT2 cells were more responsive to transformation by KRAS than general AT2 cells. Moreover, after bacterial lung injury, there was a significant increase of CD44high AT2s and niche components distributed throughout the lung parenchyma. Taken together, CD44high AT2 cells and their perivascular niche regulate tissue homeostasis and tumor formation.

Graphical abstract

Highlights

• CD44high lung alveoli type II cells (AT2s) express immunity genes in homeostasis

• Disruption of immunity gene STAT1 impairs progenitor functions of AT2 cells

• CD44high AT2s are supported by peri-macro-vessel niche in homeostasis

• CD44high AT2s and niche components expand after lung injury and in response to KRAS

Liu and colleagues characterized a CD44high subpopulation of lung alveolar type II cells (AT2s), which have high expression of immune-related genes and reside in a perivascular niche composed of LYVE1+ endothelial cells, adventitial fibroblast cells, and HA. CD44high AT2s and their niche components expand in response to lung injury. These cells are also more responsive to KRAS than bulk AT2s.

Keywords

alveoli
lung
type II cells
homeostasis
stem cell
niche
adventitial
cancer
CD44
immune signaling
Published: May 16, 2024
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pmcIntroduction

The adult human lung harbors millions of alveoli, essential for gas exchange, consisting of type I (AT1) and type II (AT2) epithelial cells (Dobbs et al., 2010; Mason, 2006; Weibel, 2009). AT1 cells, covering approximately 95% of the alveolar surface, facilitate the exchange of gases by forming an interface with the microvasculature (Dobbs et al., 2010; Weibel, 2009). In contrast, cuboidal AT2 cells are notable for surfactant production (Mason, 2006). Significantly, these cells, as a population, also act as tissue stem cells, undergoing self-renewal and differentiating into AT1 cells (Barkauskas et al., 2013; Desai et al., 2014; Evans et al., 1975). Compared with other adult tissues like the skin and intestine, lung alveolar epithelial cells exhibit low turnover rates under steady-state conditions (Barkauskas et al., 2013; Desai et al., 2014). Consequently, a limited subset of AT2 cells serves as regenerative stem cells during routine cell replenishment. Investigations into mouse and human lungs have identified molecularly distinct AT2 subsets, such as those with elevated AXIN2 levels (Nabhan et al., 2018; Travaglini et al., 2020; Zacharias et al., 2018). However, the precise roles of these subsets remain partly understood. For therapeutic applications, pinpointing specific surface markers to isolate and enrich AT2 subsets critical for regeneration is imperative. Notably, a subset of AT2 cells in uninjured mouse lungs, characterized by higher CD44 expression—a transmembrane receptor for hyaluronan (HA) in the extracellular matrix (Jiang et al., 2011)—emerges as potential stem cells for maintaining homeostasis (Chen et al., 2017). These CD44high AT2s, representing about 3% of the AT2 population, exhibit enhanced proliferative capacity and differentiation potential into AT1 cells in vitro compared to their CD44low counterparts (Chen et al., 2017).

All stem cells, including AT2 stem cells, are influenced by their local microenvironment or niche (Morrison and Spradling, 2008). For AT2 cells, this niche likely includes endothelial cells (ECs) and specific fibroblast subgroups. Lung microvascular ECs are known to affect AT2 progenitor functions post-injury through the secretion of molecules like MMP14 (Ding et al., 2011), TSP1 (Lee et al., 2014), HGF (Cao et al., 2017), and the bioactive lipid S1P (Chen et al., 2020). Similarly, certain fibroblast subgroups exhibit niche properties (Barkauskas et al., 2013; Zepp et al., 2017). Yet, the precise anatomical relationships between these stromal cells and AT2 subpopulations, particularly under basal conditions, are not clear. Here, our studies reveal that that CD44high AT2s tend to be situated near macro blood vessels, supported by a previously unidentified perivascular niche. This niche consists of LYVE1+ subgroup of non-lymphatic ECs, adventitial fibroblasts, and HA deposits. Following inflammatory lung injury, we observed an increase in adventitial fibroblast-like cells, HA, and CD44high AT2 cells across the lung parenchyma. Notably, CD44high AT2 cells uniquely express intrinsic immunity-related signaling molecules, even in the absence of apparent injury or inflammation. Significantly, we demonstrated that STAT1, one of those immune signaling mediators, is vital for the stem cell function of these AT2 cells during homeostasis. Furthermore, we discovered that in the HA-rich peri-macro-vessel microenvironment, the CD44high AT2s are particularly susceptible to KRAS-mediated transformation.

Results

The CD44high subpopulation of AT2 stem cells in both the mouse and human lung expresses a unique signature of immunity-related genes

To characterize the CD44high putative alveolar stem cell subpopulation (Chen et al., 2017) through RNA sequencing (RNA-seq) analysis, CD44high and CD44low AT2 cells were isolated from adult, uninjured mice for comparison (Figures 1A and S1A, and Table S1). CD44high AT2s exhibited gene expression profiles that distinguish them from the general AT2 population (Figures 1B and S1B). Compared with CD44low AT2s, 1,664 genes exhibited differential expression (FDR adjusted p value <0.05) in CD44high AT2s by at least 1.5-fold; 1,082 of these were upregulated, and 582 were downregulated (Figure 1B). Notably, genes associated with cell proliferation were predominantly upregulated in CD44high AT2s (Figure S1C, Table S2), though the expression of the epithelial cell marker, Cdh1 (Chignalia et al., 2015), remained consistent with the general AT2 population (Table S2). Conversely, the expression of most AT2-specific genes, including Sftpc, Sftpb, and Abac3 (Finn et al., 2019), was reduced in CD44high AT2s, while AT1 marker (Finn et al., 2019) expression was comparable between CD44high and CD44low AT2s (Figure S1D, Table S2). These data suggest that CD44high AT2s are more proliferative yet less differentiated.Figure 1 The CD44high (or CD44hi) subpopulation of AT2 stem cells in mouse lung express a unique signature of immunity-related genes

(A) CD44hi and CD44low (CD44lo) AT2s were isolated for RNA-seq studies.

(B) Differentially expressed genes (DEGs).

(C) Pathway analysis for DEGs revealed that many genes involved in immunomodulation were upregulated in CD44high AT2s.

(D) A heatmap of DEGs that are involved in inflammatory response. 3 replicates from a total of 8 mice.

(E) qPCR of immune-related genes Il1β, Tnf, Tnfrsf1b, Traf2, Il6, Ccl20, and Neurl3 in CD44hi and CD44lo AT2s. Each dot = 1 mouse. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001; Mean ± SEM. (F and G) scRNA-seq.

(F) UMAP plot to compare the distribution of isolated CD44high AT2s and total AT2s in clusters 0–4.

(G) Dotplot analysis of combined CD44high AT2s and total AT2s in clusters 0–4. Cluster 3 and 4 are proliferating cells enriched with Mki67 and Top2a. CD44 expression is enriched in cells of cluster 2 and the two proliferating clusters. 4 examples of the immunity-related genes showed higher expression in clusters enriched with CD44high AT2s. Data from pooled 6 mice. See also “Extent technical descriptions” and Figure S1, Table S1.

Further, we contrasted CD44high AT2s with the AXIN2+ AT2 stem cell subpopulation previously described (Nabhan et al., 2018; Zacharias et al., 2018) using Axin2-CreER/Tomato mice. Our analysis revealed that CD44high AT2s are not preferentially found within the EPCAM+Axin2+ AT2 subset (Figure S1E). Moreover, CD44high AT2s lack heightened expression of WNT-responsive (Dost et al., 2020) and other genes characteristic of AXIN2+ AT2s, such as Axin2 and Tm4sf1 (Zacharias et al., 2018) (Figure S1F). This delineates CD44high AT2s and AXIN2+ AT2s as molecularly distinct subpopulations.

Further investigations into signaling mechanisms influencing CD44high AT2s revealed significant upregulation of genes related to inflammation and immune regulation (Figure 1C). We conducted a heatmap analysis of differentially expressed genes within the Gene Ontology categories for positive regulation of the inflammatory response, finding most to be upregulated in CD44high versus CD44low AT2s (Figure 1D). This pattern of upregulation was absent in AXIN2+ AT2s (Figure S1G). Quantitative PCR confirmed higher expression levels of inflammatory markers, including Il-1β, Tnf, Tnfrsf1b, Traf2a, Il-6, Ccl20, and Neurl3 in CD44high AT2s (Figure 1E).

Next, we utilized isolated CD44high AT2s and total AT2 cells for single-cell RNA-seq (scRNA-seq), with the results visualized via uniform manifold approximation and projection (UMAP). Rather than forming a unique cluster, CD44high AT2s were dispersed across five distinct clusters but distributed in a manner divergent from the overall AT2 population (Figures 1F and S1H). In particular, enrichment of CD44high AT2s was observed in cluster 2 and in two clusters identified for their proliferative cell profiles (clusters 3 and 4). Aligning with findings from bulk RNA-seq, these CD44high AT2-enriched clusters exhibited elevated expression of several immunity-related genes, including NF-κB2 (Chawla et al., 2021) and Thbs1 (Mir et al., 2015) (Figure 1G). This result also shows that the CD44high AT2s in basal homeostasis are different from the regeneration-associated AT2/AT1 transitional cells, which are characterized by distinct clustering in scRNA-seq analyses (Choi et al., 2020; Kobayashi et al., 2020; Riemondy et al., 2019; Strunz et al., 2020). Further analysis of published scRNA-seq datasets from uninjured adult mice (Wang et al., 2018) corroborated our findings: while some AT2s express a higher level of CD44, they do not form distinct clusters (Figure S1I). Moreover, our RNA-seq data revealed similar Il1r1 expression levels between CD44high and CD44low AT2s (Table S1), indicating a difference from the recently characterized IL1R1+ AT2 cells (Choi et al., 2020). Collectively, these results position CD44high AT2s as a unique AT2 stem cell subset, potentially regulated by immunity-related signals.

To explore potential species-specific differences, we analyzed AT2s from human lung tissue (Figures 2A and S1J). qPCR indicated that human CD44high AT2s exhibit significantly higher expression of proliferation and immunity markers, including MKI67, TNF, TNFRSF1B, and IL-1β, compared to CD44low AT2s (Figure 2B). Using 3D organoid culture, we demonstrated that human CD44high AT2s form more and larger organoids than CD44low counterparts, mirroring observations in mice (Figures 2C and 2D) (Chen et al., 2017). Analysis of a human AT2 scRNA-seq dataset (Travaglini et al., 2020) confirmed the presence of CD44high AT2s with a distinct distribution pattern from bulk AT2s, though they did not form a separate cluster (Figure 2E). Thus, human CD44high AT2s, similar to their murine counterparts, represent a stem cell subgroup characterized by enhanced immunity signaling.Figure 2 Human CD44hi AT2s are stem cell subgroup with elevated immunity signaling

(A) FACS of human (hu) CD44hi and CD44lo AT2s.

(B) qPCR of MKI67, TNF, TNFRSF1B, and IL1β in hu CD44hi and CD44lo AT2s.

(C and D) Hu CD44hi and CD44lo AT2s in 3D organoid culture. (C) Cells were grown in colony expansion media for 2 weeks. Colony-forming efficiency and colony size were quantified. (B and C) Ratio paired t test. Each dot = 1 donor.

(D) Cells from 2 donors were further grown in differentiation media to generate AT1s. The resulting organoids were sectioned and stained for AT2 marker HTII280 and AT1 marker RAGE. Each data point represents a Matrigel drop in culture. Unpaired t test.

(E) scRNA-seq data of human AT2s were extracted from (Travaglini et al., 2020) for tSNE projection. Cells with top 30% expression of CD44 were indicated by color. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001. Mean ± SEM. Scale bars, 500 μm for C, 10 μm for D. See also Figure S1.

NF-κB and STAT1-mediated immunity-related signaling regulates the stem cell functions of CD44high AT2s in homeostasis

Phospho-P65 NF-κB, a key regulator of immunity signaling in mammalian epithelial cells (Chignalia et al., 2015; Larsen et al., 2020), is significantly more abundant in mouse CD44high AT2s compared to bulk AT2s, as shown by western blot (Figure 3A). Consistently, transcription levels of NF-κB target genes (Dost et al., 2020) are elevated in CD44high AT2s (Figure 3B). Given that IL1β is a primary activator of NF-κB signaling (Katsura et al., 2019) and that CD44high AT2s exhibit increased Il1β transcript levels (Figure 1E), we explored the impact of exogenous IL1β on CD44high AT2s using 3D mouse organoid culture. Previous studies have shown that IL1β boosts organoid formation in a co-culture system of AT2 and stromal cells, where stromal cells may partially mediate IL1β’s effects (Katsura et al., 2019). Therefore, we employed a recently developed mesenchymal-free, chemically defined organoid culture (Katsura et al., 2020). Aligning with earlier findings (Chen et al., 2017), CD44high AT2s formed significantly larger organoids than their CD44low counterparts (Figure 3C). Further segregation of CD44low AT2s into very low (CD44vl) and mid-low (CD44mi) CD44 expression levels revealed that while CD44mi AT2-derived organoids were marginally larger than those from CD44vl AT2s, the organoids originating from CD44high AT2s were considerably larger than both CD44vl and CD44mi AT2-derived organoids (Figure S2). This indicates that organoid size does not linearly correlate with CD44 expression levels and that CD44high AT2s constitute a distinct AT2 subset. Therefore our subsequent analyses concentrated on contrasting CD44high AT2s with CD44low AT2s. We observed that IL1β significantly enlarged organoids formed by both CD44high and CD44low AT2s (Figure 3C). These results collectively suggest that the IL1β-NF-κB signaling axis crucially influences the stem cell attributes of CD44high AT2s.Figure 3 NF-κB and STAT1-mediated signaling regulates CD44hi AT2s stem cell functions in homeostasis

(A) Western blot showed that CD44hi AT2s had higher levels of phospho-P65 RELA than CD44lo AT2s.

(B) A heatmap to compare DEGs between CD44hi and CD44lo AT2s that fall in “RELA targets NF-κB TRRUST”.

(C) CD44hi and CD44lo AT2s were processed for fibroblast-free organoid culture in IL1β-free control media or in media supplemented with 10 ng/mL IL1β. Tiled images resulting from stitching of adjacent areas were shown. Organoid sizes were quantified.

(D) Western blot: CD44hi AT2s express higher levels of STAT1 than CD44lo AT2s. Band intensity in A and D was quantified by ImageJ. β-ACTIN is loading control.

(E) Heatmap shows that CD44hi AT2s express higher levels of interferon response genes than CD44lo AT2s.

(F) Mouse model of tamoxifen-inducible AT2-specific knockout of STAT1.

(G) At 3 weeks after tamoxifen-induced STAT1 knockout, the mutant lungs (Stat1ΔAT2) had similar numbers of AT2s as WT (SpC-CreER/Tomato).

(H) At 8 months after tamoxifen induction, Stat1ΔAT2 lungs showed reduced number of AT2s. Lung sections in G and H were stained with AT2 lineage marker TOMATO and SP-C, and numbers of SP-C+ cells and DAPI+ cells of each lung were quantified. ∗: p < 0.05, ∗∗: p < 0.01. Each dot = 1 mouse. N ≥ 3 mice. Mean ± SEM. Scale bars, 500 μm in C and 70 μm in G and H. See also “Extent technical descriptions” and Table S1.

Another pivotal regulator of epithelial immunity signaling is the STAT family of transcription factors, with STAT1 being particularly crucial for interferon responses (Larsen et al., 2020; Naik et al., 2018; Stark and Darnell, 2012). Observations across multiple species have identified that stem cells in various tissues express interferon-stimulated genes, albeit without a clear source of interferon (Wu et al., 2018). This suggests an important role for STAT1 in tissue stem cell regulation, prompting our investigation into STAT1’s function within CD44 high AT2s. We found that CD44high AT2s express 2-fold higher levels of STAT1 compared with CD44low AT2s (Figure 3D). This increased STAT1 expression correlates with elevated levels of interferon-responsive genes in CD44high AT2s compared to CD44low AT2s (Figure 3E). To elucidate STAT1’s role in AT2s, we employed an inducible AT2-specific STAT1 knockout mouse model, Stat1ΔAT2 (Figure 3F). Given the higher STAT1 expression in CD44high AT2s, they are preferentially affected by this genetic alteration. 8 months post-STAT1 deletion, the mutant lungs displayed a significant reduction in AT2 numbers relative to wild type, which was not evident at 3 weeks post-knockout induction (Figures 3G and 3H). These findings underscore the necessity of STAT1 for maintaining AT2 cell steady-state homeostasis, particularly within the CD44high AT2 subset.

CD44high AT2s are enriched near LYVE1+ blood vessels that form an HA-high niche together with Gli1+ adventitial fibroblasts

Having identified CD44high AT2s as a unique subset distinguished by an immunoregulatory signature, we investigated their specific anatomical niche in the mouse lung. Prior research suggested these cells were predominantly located near blood vessels (Chen et al., 2017), yet the implications and precise nature of these vessels remained unclear. Recent scRNA-seq analyses differentiate ECs of macro blood vessels (diameter >50 μm) from microvasculature by markers such as von Willebrand factor (Vila Ellis et al., 2020). We found that ECs lining macro-vessels express LYVE1, an HA receptor similar to CD44 (Jiang et al., 2011) (Figures 4A, S3A, and S3B). LYVE1 is traditionally considered as a marker for lymphatic ECs (Baluk and McDonald, 2008). However, in the distal lung, LYVE1 expression is not only limited to lymphatic ECs (which are T1α+) (Baluk and McDonald, 2008) but is also detectable in the ECs of virtually all macro blood vessels (Figures S3A–S3C). This observation is consistent with earlier reports (Baluk and McDonald, 2008; Mori et al., 2013; Weber et al., 2018). Immunostaining of lung sections revealed a significant enrichment of CD44high AT2s near LYVE1+ macro-vessels compared to capillaries (Figures 4A and 4B). Additionally, 3D reconstructions from 60-μm-thick sections and whole-mount antibody staining clarified that some CD44high AT2s, seemingly distant from LYVE1+ vessels in 2D images, are in proximity to these vessels in 3D (Figures 4C–4F).Figure 4 LYVE1+ macro blood vessel ECs are niche components for CD44hi AT2s

(A–F) Immunostaining of lung section of SpC-CreER/Tomato mice. AT2s are TOMATO+. (A) CD44hi AT2s located near (arrows) or away from (arrowhead) LYVE1+ macro blood vessels are shown.

(B) ∼40% of the CD44hi AT2s were at para-LYVE1+ vessel regions (within one cell diameter) whereas only ∼10% of CD44lo AT2s are in this proximity. Mean ± SEM.

(C–F) 3D reconstruction of confocal imaging shows that CD44hi AT2s associate with LYVE1+ macro-vessels.

(C) Reconstructed z section images show the locations of LYVE1+ blood vessels and CD44hi and CD44lo AT2s in 3D. (D–F) Optimal 2D sections to show individual AT2s #1–3.

(D) Cell #1, a CD44hi AT2 does not appear to be adjacent to a LYVE1+ vessel in the 2D image but can be seen to be adjacent to a branch of a LYVE1+ blood vessel in 3D.

(E) Cell #2, a CD44hi AT2 adjacent to a LYVE1+ vessel and (F) cell #3, a CD44lo AT2 away from LYVE1+ vessel.

(G) CD44hi and CD44lo AT2s from SpC-CreER/Tomato mice were co-cultured with LYVE1+ or LYVE1− ECs for 14 days in 3D Matrigel in fibroblast-free condition. LYVE1+ ECs supported organoids formation by CD44hi and CD44lo AT2s. Organoid sizes were quantified. Box whisker plot.

(H) qPCR showed that LYVE1+ ECs expressed significantly higher levels of Fgf7 than LYVE1− ECs. Scale bar, 50 μm for A, 20 μm for C–F, 1500 μm for G. ∗: p < 0.05. ∗∗: p < 0.01; ∗∗∗: p < 0.001. N = 3–5 mice. (H) is paired t test. See also “Extent technical descriptions” and Figure S3.

In exploring the role of non-lymphatic LYVE1+ ECs on CD44high AT2 progenitor functions, we employed a mouse EC-AT2 organoid co-culture system under fibroblast-free, chemically defined conditions (Figures 4G and S3D). Compared with LYVE1− ECs, LYVE1+ ECs facilitated the formation of significantly larger organoids by both CD44high and CD44low AT2s (Figure 4G). This enhancement correlates with LYVE1+ ECs’ elevated expression of Fgf7, which encodes a factor known to promote AT2 progenitor function (Brownfield et al., 2022; Liberti et al., 2021) (Figure 4H), suggesting that LYVE1+ ECs bolster CD44high AT2 progenitor functions through paracrine signaling.

Given that both CD44 and LYVE1 are HA receptors (Jiang et al., 2011), we investigated HA’s spatial distribution in the distal lung by staining tissue sections with HA-binding protein (Tammi et al., 1998). Our findings reveal a preferential localization of HA near LYVE1+ vessels (Figure 5A), with approximately 60% of CD44high AT2s situated in proximity to HA. This represents a significant enrichment when compared to CD44low AT2s (Figure 5B), suggesting that HA constitutes a component of the CD44high AT2s niche surrounding LYVE1+ vessels. This observation aligns with previous data indicating that HA supplementation enhances AT2 organoid growth (Chen et al., 2017).Figure 5 HA and Gli1+ adventitial fibroblasts are components of the perivascular niches for CD44hi AT2s

(A, B, D, E, and H) Immunostaining of mouse lung sections.

(A) Distribution of HA (detected by HABP) is closely associated with LYVE1+ blood vessels.

(B) Lungs of SpC-CreER/Tomato mice: CD44hi AT2s are preferentially located near HA-enriched areas (arrows) compared to HA low areas (arrowheads).

(C) CD44hi or CD44lo AT2s cultured in stromal-free organoid culture, DMSO (Con), or 4MU were included.

(D–F) Gli1-CreER/Tomato lungs.

(D) The locations of Gli1+ cells and the HA accumulation are highly correlated at para-LYVE1+ vessel regions. V, vessel.

(E) CD44hi AT2s (SP-C+CD44+) are preferentially localized near Gli1+ cells (arrows).

(F) qPCR of Has1-3 expression in Gli1+ and Gli1− mesenchymal cells.

(G) Unsupervised clustering of scRNA-seq data of mouse lung fibroblasts (GEO: GSE132771). Has1 is preferentially expressed by adventitial fibroblasts defined by Col14a1 expression.

(H) Gli1-CreER/Tomato/Pdgfrα-GFP lungs. Pdgfrα+ cells do not show preferential adventitial localization, but there is overlap between these cells and Gli1+ cells. Arrowheads: Gli1+Pdgfrα+ cells. Arrows: Gli1+Pdgfrα− cells. Also note that a substantial fraction of Gli1+ cells are located near LYVE+ macro blood vessels. Scale bar, 50 μm for A, B, D, E, H; 1000 μm in C. ∗: p < 0.05; ∗∗: p < 0.01. ∗∗∗: p < 0.001. Mean ± SEM. N = 3 mice. See also “Extent technical descriptions” and Figure S5.

HA is synthesized by three HA synthases, encoded by Has1, Has2, and Has3 (Jiang et al., 2011). Analysis of our RNA-seq data indicated that CD44high AT2s express significantly higher levels of Has3 compared to the general AT2s (Table S1). In line with this, treatment with the HA synthase inhibitor 4-methylumbelliferone (4MU) (Marshall et al., 2021) resulted in a significant reduction in the size of organoids formed by CD44high AT2s in our stromal-free culture (Figure 5C).

In addition to ECs, fibroblasts and immune cells have been posited to contribute to the niche that supports AT2 stem cells (Juul et al., 2020; Wu and Tang, 2021). Thus, our investigation extended to other stromal cells preferentially co-localizing with CD44high AT2s near macro blood vessels. Ng2+ pericytes (Crisan et al., 2008) did not demonstrate a specific proximity to CD44high AT2s (Figure S4A), indicating they might not be integral to this niche. Similarly, CD44high AT2s did not exhibit a preferential localization near CD45+ immune cells (Hermiston et al., 2003) (Figure S4B), suggesting that these immune cells may not play a supportive role in the basal homeostasis of CD44high AT2s.

Our focus then shifted to adventitial fibroblasts, a subset of fibroblasts recently characterized by scRNA-seq as being closely associated with adventitial cuffs and blood vessels in the distal lung (Tsukui et al., 2020). We found that in addition to the known adventitial fibroblast markers like COL14A1 (Tsukui et al., 2020), Gli1 is also preferentially expressed by this subgroup of fibroblasts (Figures S5A and S5B). A significant portion of Gli1+ cells was found in regions adjacent to macro blood vessels and HA (Figures 5D and S5C). In addition, nearly 60% of CD44high AT2s were located near Gli1+ cells, as opposed to about 20% of CD44low AT2s (Figure 5E). Furthermore, qPCR showed that Gli1+ mesenchymal cells exhibited significantly higher expression levels of Has1 and Has2 than their Gli1− counterparts (Figures S5D and 5F). Analysis of the published scRNA-seq dataset (Tsukui et al., 2020) confirmed the preferential expression of Has1 in adventitial fibroblasts (Figure 5G).

Given the recognized role of PDGFRα+ (Barkauskas et al., 2013) and AXIN2+PDGFRα+ (Zepp et al., 2017) fibroblast subsets in forming a niche for AT2s, we examined the relationship between Gli1+ cells and these cells. We observed an overlap between Gli1+ cells and both subsets (Figures 5H, S5E, and S5F), further supporting the notion that Gli1+ adventitial fibroblasts, through their production of HA, are a crucial component of the HA-rich peri-LYVE1+ vascular niche supporting the CD44high AT2s.

Inflammatory injury results in a concomitant increase of CD44high AT2 cells and their niche

To determine the responses of CD44high AT2 cells and their niche components after lung injury, we utilized a Pseudomonas aeruginosa (PA)-induced mouse lung injury model (Finn et al., 2019). Three days post-PA infection, coinciding with the early repair phase (Finn et al., 2019), there was a noteworthy expansion of COL14A1+ adventitial fibroblast-like cells throughout the lung, whereas the distributions of LYVE1+ blood vessels were not changed (Figure 6A). At this period, there was also a substantial upsurge in HA accumulation, alongside a significant increase in the proportion of CD44high AT2s among the total AT2 cell population (Figures 6B and 6C). Earlier research indicated that a subset of AT2 cells expressing the stem cell marker SCA-1 emerges during the repair phase post-PA lung injury (Liu et al., 2015). Here we found that nearly all post-PA CD44high AT2s were SCA-1+ (Figure 6C), diverging from basal state CD44high AT2s, which are SCA-1− (Chen et al., 2017).Figure 6 Increase of COL14A1+ adventitial fibroblast-like cells, HA, and CD44hi AT2s after bacterial lung injury

(A and B) Mice lung sections were prepared for immunostaining at 3 days after PA i.t. (intratracheal injection) and compared with non-PA controls.

(A) After PA, the amount of COL14A1+ adventitial fibroblast-like cells was significantly increased, and these cells were distributed throughout the lung parenchyma. However, the distributions of LYVE1+ cells were not changed as most LYVE1+ cells lined macro-vessels (arrowheads).

(B) The amount of HA was significantly increased post PA as detected by HABP. The enlarged areas show distributions of CD44hi AT2s (arrows). The percentage of CD44hi AT2s vs. total AT2s was significantly increased with PA treatment. Scale bar, 50 μm.

(C) FACS of CD44hi AT2s at 3 days PA. Most CD44hi AT2s also express another stem cell marker SCA-1. The percentage of CD44hi AT2s vs. total AT2s was quantified.

(D) Comparison of transcriptome profile between basal state CD44hi AT2s and post-PA CD44hi AT2s. Heatmaps were generated from RNA-seq data of 4 groups of cells: basal state CD44hi AT2s, 3 days post-PA CD44loSCA-1−, 3 days PA CD44loSCA-1+, and 3 days PA CD44hi AT2s. The following gene sets were compared: (1) Cell proliferation genes. (2) AT2 markers. (3) Markers for transitional AT2/AT1 intermediate cells (DATP/PATS), defined by (Choi et al., 2020; Kobayashi et al., 2020). (4) Inflammatory response genes. (5) Interferon response genes. ∗∗∗: p < 0.001. Each dot = 1 mouse. Mean ± SEM. N ≥ 3 mice. Also see “Extent technical descriptions” and Table S3.

To further compare basal state CD44high AT2s and post-PA CD44high AT2s, we performed RNA-seq using AT2s of non-PA or 3 days post-PA lungs. Post-PA CD44high AT2s exhibit an enhanced expression profile of cell proliferation markers, a reduction in AT2 markers, and a significant elevation in markers for transitional AT2/AT1 cells (Choi et al., 2020; Kobayashi et al., 2020) compared to the basal state CD44high AT2s and post-PA CD44low AT2s (Figure 6D). These characteristics suggest post-PA CD44high AT2s are involved in lung repair and enriched with AT2/AT1 intermediate cells. Moreover, many immunity-related transcripts were further upregulated in post-PA CD44high AT2s compared with the basal state CD44high AT2s (Figure 6D). Taken together, inflammatory lung injury induces a synergistic increase in CD44high AT2s, COL14A1+ adventitial fibroblast-like cells, and HA deposition throughout the lung parenchyma. The concurrent accumulation of CD44high AT2s alongside niche components indicates a pivotal role for this AT2 stem cell-niche system in the repair process following injury.

CD44high AT2s in the HA-high perivascular niche are more susceptible to KRAS tumor induction

AT2s are a major source of cells that undergo KRAS-induced transformation leading to lung adenocarcinoma (Dost et al., 2020). Early after KRAS induction, a subset of AT2s exhibit NF-κB pathway activation and partial dedifferentiation (Dost et al., 2020), characteristics notably shared by CD44high AT2s (Figure 3A, 3B, and S1D). Furthermore, CD44high AT2s express higher levels of genes typical of early-stage adenocarcinoma (Neidler et al., 2019) (Figure 7A) or those transcripts enriched in AT2 subpopulations responsive to KRASG12D (Figures S6A, S6C, S6E, S6G, and S6H), differing from CD44low AT2s, whose transcriptome aligns with AT2 subsets unresponsive to KRASG12D (Figures S6B, S6D, and S6F) (Dost et al., 2020) (Table S4).Figure 7 CD44hi AT2s in their niche are responsive to KRAS tumor induction

(A) CD44hi AT2s express higher levels of lung adenocarcinoma (LUAD) signature genes (Neidler et al., 2019) than CD44lo AT2s as revealed by a heatmap of DEGs between CD44hi and CD44lo AT2s.

(B) AT2 lineage tracing SpC-CreER/Tomato mice were treated with urethane. COL14A1+ cells, LYVE1+ vessels (arrows), and TOMATO+ AT2 lineage-labeled flat AT1-like cells (arrowheads) were associated with AT2 hyperplasia foci at 12 weeks post treatment.

(C) CD44hi AT2s were more responsive to KRAS than CD44lo AT2s in vitro and in vivo as shown using a KrasG12D/Tomato/SpC-GFP LUAD mouse model. KRASG12D expressing CD44hi and CD44lo AT2s were grown in 3D culture or were transplanted into mouse lungs that were injured with bleomycin 24 h earlier. The lungs were examined 8 weeks after transplantation. The areas with TOMATO versus total lung areas were quantified. Scale bar, 50 μm in B, 500 μm in C-upper panel, 3 mm in C-lower panel. ∗: p < 0.05. Each dot = 1 mouse. Mean ± SEM. N ≥ 3 mice. See also “Extent technical descriptions”, Figures S6 and S7, and Table S4.

(D) A diagram depicting the preferential localization of CD44hi AT2 stem cells near macro blood vessels lined by LYVE1+ ECs, in contrast to CD44lo AT2s found near LYVE1− microvascular ECs. CD44hi AT2s exhibit intrinsic immunity-related signaling, crucial for their stem cell functions in steady state. The LYVE1+ ECs and adjacent adventitial fibroblasts constitute a perivascular niche, supporting CD44hi AT2s through paracrine factors (blue arrow) and HA deposition (brown arrow). Following inflammatory lung injury, expansions of CD44hi AT2s, adventitial fibroblast-like cells, and HA deposition occur. Notably, CD44hi AT2s in this perivascular niche show heightened responsiveness to KRAS-driven adenocarcinoma induction compared to CD44lo AT2s.

In exploring whether CD44high AT2s in distinct perivascular niches are more likely to evolve into pre-adenocarcinoma cells than other AT2s, we utilized SpC-CreER/Tomato mice treated with urethane, which activates the RAS pathway and induces lung adenocarcinoma in approximately one year (Li et al., 2020). By 12–14 weeks post-treatment, we observed numerous AT2 hyperplastic foci containing CD44high AT2s and many AT2 lineage-labeled flat AT1-like cells (Figures 7B, S6I, and S6J). This suggests AT2s within these foci are proliferative and inclined toward AT1 differentiation, a propensity akin to CD44high AT2s (Chen et al., 2017). Notably, these foci were often associated with LYVE1+ vessels, showing a significantly increased presence of COL14A+ adventitial fibroblast-like cells and HA deposition (Figures 7B, S6I, and S6J).

Since early-stage or pre-adenocarcinoma cells manifest transcriptome and phenotypic similarities with CD44high AT2s and reside in a similar HA-high niche with adventitial fibroblast-like cells and LYVE1+ vessels, we assessed CD44high AT2s’ responsiveness to RAS activation in a modified KrasG12D mouse and organoid setup (Dost et al., 2020) (Figure 7C). On day 5 after KRASG12D induction, the colonies formed by CD44high AT2s were significantly larger than those by CD44low AT2s (Figure 7C); cells in KRAS-activated colonies appeared partly dedifferentiated and lost most SpC-GFP expression (Figures S7A and S7B). Interestingly, CD44low AT2s also responded to KRASG12D by first becoming CD44+ and later expanding and differentiating (Figures S7A–S7C). To evaluate the in vivo responsiveness of CD44high AT2s to KRAS, CD44high and CD44low AT2s from KrasG12D/Tomato/SpC-GFP mice were treated with adenovirus expressing Cre and transplanted into bleomycin-pre-injured lungs (Dost et al., 2020) (Figure 7C). Eight weeks post-transplantation, lungs receiving CD44high AT2s developed significantly more tumors than those with CD44low AT2s (Figures 7C and S7D–S7F). Thus, CD44high AT2s are more susceptible to KRAS induction than bulk AT2s both in vitro and in vivo.

Discussion

In this study, we have characterized a subset of CD44high AT2s, which are preferentially localized near macro blood vessels, distinguishing them as a unique group of AT2 stem cells during steady-state homeostasis (Figure 7D). The CD44high AT2s in basal homeostasis are distinct from previously identified lung epithelial stem cells — such as bronchioalveolar stem cells, lineage-negative epithelial progenitors, and AXIN2+ AT2 subpopulations (Chen et al., 2017; Kim et al., 2005; Nabhan et al., 2018; Vaughan et al., 2015; Zacharias et al., 2018; Zuo et al., 2015). Moreover, they are distinct from regeneration-associated AT2 transitional cells and IL1R1+ AT2s (Choi et al., 2020).

Intriguingly, homeostatic CD44high AT2s display an increased expression of immunity-related transcripts in the absence of apparent injury, inflammation, or infection. Interestingly, we observed no corresponding increase in endogenous CD45+ immune cells near CD44hi AT2s. This suggests that, unlike previously characterized tissue stem cells activated by immune cells post-damage (Choi et al., 2020; Karin and Clevers, 2016; Katsura et al., 2019; Naik et al., 2018), CD44high AT2s inherently engage in immunity signaling. Crucially, we demonstrated that immunity signaling mediated by transcription factors such as NF-κB and STAT1 plays an important role in regulating the stem cell functions of CD44high AT2s under homeostatic conditions. A similar subset of CD44high AT2 stem cells that express immunity-related genes are also observed in human lungs, suggesting a conserved yet underexplored mechanism of stem cell maintenance and function in mammalian lungs.

CD44high AT2s are notably situated near macro blood vessels, lined by LYVE1+ ECs, which significantly enhance the growth of CD44high AT2-derived organoids compared to LYVE1− ECs. This enhancement is likely attributed to the higher expression of FGF7 and other angiocrine factors by LYVE1+ ECs. While previous studies have emphasized the role of microvascular ECs in releasing angiocrine factors to support alveolar epithelial cell regeneration post-injury (Cao et al., 2017; Chen et al., 2020; Ding et al., 2011; Lee et al., 2014; Niethamer et al., 2020; Vila Ellis et al., 2020), the function of macro-vessel ECs as angiocrine niches remains largely unexplored. Our findings reveal that LYVE1+ macro-vessel ECs act as pivotal niche components, regulating a subset of alveolar epithelial stem cells during basal homeostasis.

This study highlights the significance of perivascular regions enriched in HA around CD44high AT2s within the distal lung. Given that CD44 functions as an HA receptor, interactions between HA and CD44 may play a role in adhesive interactions and the stabilization of paracrine factor gradients (Zoller, 2011), and thus are crucial for the homing and retention of CD44high AT2 stem cells in their niches. Our findings suggest CD44high AT2s and Gli1+ adventitial fibroblasts as the primary sources of HA accumulation in these niches, attributed to their expression of HAS enzymes. Moreover, organoid studies confirm the necessity of HA for the stem cell functionality of CD44high AT2s. Gli1+ adventitial fibroblasts exhibit partial overlap with PDGFRα+ and AXIN2+ fibroblast subsets, previously identified as niche cells for AT2s (Barkauskas et al., 2013; Zepp et al., 2017), indicating a complex fibroblast heterogeneity within the niche. Beyond HA production, Gli1+ cells may release paracrine signals supporting CD44high AT2s. Investigating how perivascular components influence CD44high AT2s expression of immune-related signaling molecules, which regulate their stem cell functions, remains an essential future inquiry.

In response to bacterial lung injury, we observed a coordinated expansion of CD44high AT2s and their niche components, including COL14A+ mesenchymal cells and HA, without a concurrent expansion of LYVE1+ vessels. This suggests that the expansion is supported by existing lung microvascular ECs, known to express elevated angiocrine factors post-injury (Cao et al., 2017; Chen et al., 2020). Post-injury lung microvascular ECs comprise subpopulations with distinct gene expression profiles (Zhang et al., 2022), suggesting that particular EC subsets play a role in regulating CD44high AT2s after injury.

Although it is not clear whether all the CD44high AT2s present after PA lung injury are derived from pre-existing CD44high AT2s present in uninjured lung, our data strongly indicate a synergistic response between CD44high AT2s and their niche components following lung injury. Transcriptomic analysis revealed that post-injury CD44high AT2s are enriched with regeneration-associated AT2/AT1 transitional cells (Kobayashi et al., 2020), implicating their involvement in alveolar repair. The observed increase in adventitial-like fibroblasts and HA likely supports CD44high AT2 stem cell function and facilitates stem cell activation during the repair process (Nakka et al., 2022).

One of the broader implications of our work is that the perivascular stem cell-niche microecosystem may have additional functions, e.g., in the context of endogenous tumor growth. Notably, CD44high AT2s exhibit heightened responsiveness to KRAS activation compared to their bulk AT2 counterparts. Urethane-induced hyperplastic foci in the lung are associated with COL14A1+ adventitial fibroblast-like cells, HA, and LYVE1+ vessels. This suggests that the expansion of CD44high AT2s — or the reprogramming of CD44low AT2s into CD44high AT2-like cells within this niche — may be crucial during the initial stages of preneoplastic transformation.

In summary, our findings underscore that high CD44 expression marks a subset of AT2s residing in a progenitor state within a perivascular niche, with LYVE1+ ECs, associated adventitial fibroblasts, and HA constituting critical niche components. These CD44high AT2s regulated by intrinsic immunity-related signaling appear central to homeostatic maintenance and primed for activation upon lung injury to participate in the subsequent repair process. This primed state is presumably sustained by the perivascular niche. Given their proximity to blood vessels—sites of heightened pathogen exposure—these cells may serve a sentinel function, rapidly initiating regenerative responses upon insult detection. Moreover, our in vivo studies demonstrating increased responsiveness of CD44high AT2s to KRAS underscore their stem cell characteristics and establish a connection between inflammation and cancer. Importantly, we observed integrated responses from both CD44high AT2s and niche components during post-injury repair and lung adenocarcinoma initiation, indicating that CD44high AT2s and the perivascular niche collectively function as a regenerative hub. This hub is essential for maintaining alveolar homeostasis under basal conditions, during injury response, and its disturbance may contribute to tumor development (Figure 7D).

Experimental procedures

Resources availability

Lead contact

Further information and requests for reagents may be directed to and will be fulfilled by Dr. Yuru Liu (yuruliu@uic.edu).

Material availability

This study did not generate new unique reagents.

Data and code availability

• The accession number of bulk RNA-seq of CD44high and CD44low AT2s reported in this paper is GEO: GSE182699 and GEO: GSE183152. The accession number of raw and processed data of scRNA-seq of CD44high and total AT2s is GEO :GSE202226. The data are publicly available as of the date of publication.

• Code for AT2 cell scRNA-seq analysis is available at: https://github.com/LiulabAlveolar/CD44.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Animal care and use

Both male and female mice were used. See Table S5 and “supplemental experimental procedures” for mouse lines, tamoxifen, PA, and bleomycin administration.

Isolation of mouse and human CD44high and CD44low AT2s

AT2s were isolated by fluorescence-activated cell sorting (FACS) using lineage marker (mouse) or HTII280 (human) from dissociated lung single-cell suspension and further separated by CD44 expression levels. See Table S5 and “supplemental experimental procedures” for details.

Isolation of LYVE1+ and LYVE1− ECs, Gli1+ and Gli1− mesenchymal cells

Mouse lungs were dissociated into single-cell suspensions and proceeded to FACS. For ECs, DAPI−EPCAM−CD45−T1α− cells were further separated into CD31+LYVE1+ and CD31+LYVE1− populations. For fibroblasts, CD45−CD31−EPCAM− cells from Gli1-CreER/Tomato mice were further separated into TOMATO+ and TOMATO−. See also Table S5 and “supplemental experimental procedures.”

Antibody staining

Mouse lungs were processed for paraffin embedding, sectioning, and staining. For some experiments, lungs were fixed, embedded in OCT, and cut into 60-μm-thick section for whole-mount staining. Images were captured using confocal microscope. See Table S5 and “supplemental experimental procedures” for details and for image quantification methods.

Western blot and quantitative reverse-transcription PCR

Freshly isolated lung cells were used. See Table S5 and “supplemental experimental procedures” for antibodies used, PCR primers, and details.

3D organoid culture of mouse and human AT2s

AT2s were cultured in Matrigel in chemically defined fibroblast-free conditions (Katsura et al., 2020). In some cases, ECs, IL1β, and 4MU were included. See Table S5 and “supplemental experimental procedures” for details and media components.

Mouse lung adenocarcinoma models

For the urethane model, urethane dissolved in normal saline was intraperitoneally injected into SpC-CreER/Tomato mice (C57BL/6 background) at 1 mg/g (body weight). The injection was repeated weekly for a total of 6 times.

For KRASG12D lung adenocarcinoma model, AT2s were isolated from KrasG12D/Tomato/SpC-GFP mice and treated with adenoviral-Cre; and proceed for organoid culture or transplantation. See Table S5 and “supplemental experimental procedures” for details.

Population and scRNA-seq analysis

CD44high and CD44low AT2s were collected by FACS from lungs of adult AT2 lineage labeling mice and processed for RNA-seq at the Northwestern University NUseq core. Sequencing data were processed for PCA, pathway, heatmap, and UMAP analysis.

Some published scRNA-seq datasets about mouse lung AT2s, ECs, fibroblasts, and human AT2s were downloaded from GEO or Synapse and analyzed using Seurat package in R. See Table S5 and “supplemental experimental procedures” for details.

Statistics

Data are presented as mean ± SEM. Student’s t test was used to determine statistical significance. Unpaired t test was performed unless otherwise specified in figure legends. p < 0.05 is considered as significant. Each dot in bar graphs represents one animal unless otherwise stated in figure legends.

Study approval

All animal studies were approved by the Institutional Animal Care Committee of the University of Illinois at Chicago (UIC). The human lungs were procured under University of North Carolina Institutional Review Board (IRB)-approved protocols. Tissues were provided to UIC with basic demographic information, but without personal identifying information. This was determined as “not human subjects research” by IRB of UIC.

Supplemental information

Document S1. Figures S1–S7 and supplemental experimental procedures

Table S1. Inflammation markers for Figures 1 and 3

Table S2. Additional AT2 genes for Figure S1

Table S3. Post-injury AT2 genes for Figure 5D

Table S4. KRAS response genes for Figures 7A and S6

Table S5. Reagent or resource for experimental procedures

Document S2. Article plus supplemental information

Acknowledgments

We thank Dr. Brigid Hogan (Duke University) for discussion and comments and Jiaxin An and Emilio Edemni for technical assistance. This work was supported by 10.13039/100000002 NIH grants R01HL105947 (Y.L.) and R01HL155272 (Y.L.), P01HL160469 (J.R.), R01HL163978 (J.R.), ALA-IA691074 (Y.L.), AHA-19POST34380566 (Q.C.), ATS-unrestricted grant (Q.C.), 10.13039/100000002 NIH -1S10OD025120 (NUseq) and CF Foundation grant BOUCHE19R0 and NIH DK065988 (S.H.R.).

Author contributions

Conceptualization, Y.L., Q.C., and J.R.; methodology, Y.L., Q.C., H.H., M.C., M.C., J.Z., S.H.R., and P.K.L.M.; investigation, Y.L., Q.C., H.H., M.C., M.C., J.Z., P.K.L.M., and J.R.; writing – original draft, Q.C. and H.H.; writing – review and editing, Y.L. and J.R.; funding acquisition, Y.L., J.R., Q.C., and S.H.R.; resources, Y.L.; supervision, Y.L.

Declaration of interests

The authors declare no competing interests.

Declaration of AI-assisted technologies in the writing process

During the preparation of this work, the author used ChatGPT4.0 to improve readability and language. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Supplemental information can be found online at https://doi.org/10.1016/j.stemcr.2024.04.009.
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