Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor

Abstract

Functional tissue regeneration is required for the restoration of normal organ homeostasis after severe injury. Some organs, such as the intestine, harbour active stem cells throughout homeostasis and regeneration1; more quiescent organs, such as the lung, often contain facultative progenitor cells that are recruited after injury to participate in regeneration2,3. Here we show that a Wnt-responsive alveolar epithelial progenitor (AEP) lineage within the alveolar type 2 cell population acts as a major facultative progenitor cell in the distal lung. AEPs are a stable lineage during alveolar homeostasis but expand rapidly to regenerate a large proportion of the alveolar epithelium after acute lung injury. AEPs exhibit a distinct transcriptome, epigenome and functional phenotype and respond specifically to Wnt and Fgf signalling. In contrast to other proposed lung progenitor cells, human AEPs can be directly isolated by expression of the conserved cell surface marker TM4SF1, and act as functional human alveolar epithelial progenitor cells in 3D organoids. Our results identify the AEP lineage as an evolutionarily conserved alveolar progenitor that represents a new target for human lung regeneration strategies.

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Figure 1: Identification of an Axin2+ AEP in the adult lung that regenerates a substantial percentage of the alveolar epithelium.
Figure 2: AEPs possess a distinct transcriptome and chromatin architecture enriched for cell-cycle and progenitor-cell pathways.
Figure 3: Identification of Tm4sf1 as an AEP-specific cell surface marker capable of isolating functional human AEPs.
Figure 4: AEPs display an evolutionarily conserved response to Wnt and Fgf signalling.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (T32-HL007586 to W.J.Z; T32-HL007915, K12-HD043245 to D.B.F., T32-HL007843 to J.A.Z. and HL110942, HL087825, HL132999, HL129478, HL134745 to E.E.M.). We thank the Flow Cytometry Core Laboratory of Children’s Hospital of Philadelphia and the CVI Histology Core, Next Generation Sequencing Core and CDB Microscopy Core at the University of Pennsylvania for technical assistance.

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Contributions

W.J.Z., D.B.F., J.A.Z., F.A.A., S.Z. and J.K. performed the experiments. W.J.Z., D.B.F., J.A.Z., M.P.M. and E.E.M. analysed the data. E.C. provided access to human samples and assisted W.J.Z. with all human experiments. E.E.M. supervised the project. W.J.Z. wrote the first draft of the manuscript. All authors contributed to the writing of the final manuscript.

Corresponding author

Correspondence to Edward E. Morrisey.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks C. Dean and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Location of Axin2+ epithelial cells within the adult mouse lung.

a, Low-power view of the lung showing that E-cadherin+Axin2+ epithelial cells are found only in the alveolar region, and not in the airway of the lung. b, c, Immunohistochemistry for ciliated (b) and secretory (c) markers shows no evidence of Axin2-lineage labelled cells co-expressing either of these markers. d, e, Quantification of the location of Axin2+ epithelial cell distribution in the lung. f, qPCR showing that Axin2+ AEPs and AT2 cells express similar levels of AT2 markers and other lung epithelial cell markers. AEPs express slightly higher levels of Abca3. g, AEPs express increased levels of Wnt signalling pathway components and targets by qPCR. hj, Cytopsins and quantification demonstrating that the majority of sorted Axin2+ epithelial cells are Sftpc+. k, l, FACS analysis of Axin2tdT-positive, HopxeYFP mice demonstrating that few Axin2+ epithelial cells express Hopx, consistent with the immunohistochemistry data shown in Fig. 1. Data in this figure represent n = 3 (k, l), 4 (dj) or 10 (all other panels) mice from three individual experiments. Statistics are representative of all biological replicates. All data are shown as centred on mean with bars indicating standard deviation. *P < 0.05, **P < 0.01 by two-tailed t-test (f, g) or ANOVA with preplanned pairwise comparisons and adjustment for multiple comparison testing (d). Scale bars: ac, 100 μm; h, i, 25 μm. Source data

Extended Data Figure 2 Characterization of Axin2+ Wnt responsive cells in the adult lung.

a, Lineage tracing for three months shows a stable population of AEPs and progeny in the alveolar epithelium. Yellow arrow, labelled cell; white arrow, unlabelled cell. b, c, Quantification of AT1 and AT2 cells labelled by the AEP lineage mark at homeostasis. Lower power (df) and higher power (gi) images showing expansion of AEPs in a regional fashion, one month after influenza injury. Dotted white line in f shows the edge of a Krt5+ pod, with a dearth of AEP-lineage-labelled cells. Panels gi show additional channels of the same fields as shown in Fig. 1i, j. j, Representative FACS plot showing expansion of AEP-lineage-labelled epithelial cells after influenza. The quantification of these FACS plots can be found in Fig. 1n. ko, Comparison of Ki67+ expression in AT2 cells and AEPs after influenza. In areas of regeneration, Ki67+ AEPs constitute the majority of cells entering the cell cycle, when compared to AT2 cells. Data shown represent n = 5 (jo), 6 (ac) or 10 (di) independent mice from three individual experiments, except for the nine-month lineage tracing which was performed in two separate experiments. Statistics are representative of all biological replicates. All data are shown as centred on mean with bars indicating standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 by ANOVA with preplanned pairwise comparisons and adjustment for multiple comparison testing. Scale bar, 50 μm. Source data

Extended Data Figure 3 In contrast to adult lung homeostasis, the Wnt response in the alveolar epithelium during alveologenesis is dynamic.

a, Schematic of lineage labelling procedure to assess Wnt-responsive epithelium during alveologenesis. b, Epithelial cells were identified by FACS as Epcam+CD45CD31. Cells were then gated for tdTomato and eYFP expression as shown. c, Quantification of Wnt responsiveness in the alveolar epithelium over a 1-day or 3-week lineage trace. d, Model of directionality and magnitude of AT2 and AEP transitions. During alveologenesis, AT2 and AEP fates are somewhat fluid, though the AEP population decreases during this period of lung development. During adult homeostasis, few if any AT2 cells take on the AEP fate (see Fig. 2). After injury, AEPs expand to create AT2 cells, but even after injury very few AT2 cells adopt the AEP fate. Data shown represent n = 3 mice. Statistics are representative of all biological replicates. Data in c are centred on mean with bars indicating standard error of the mean. Source data

Extended Data Figure 4 AEPs are a distinct lineage compared to Sox2-derived Krt5+ cells and are capable of generating AT1 cells.

ad, AEPs and Krt5+ cells inhabit distinct regions of the regenerating mouse lung. a, Overview of a region surrounding a Krt5+ pod. b, In regions of mild injury, AEPs and AEP-lineage-marked AT2 cells predominate and no Krt5+ cells are seen. Yellow arrow, AEP-labelled cell. c, At the border of zone 4 areas of alveolar destruction, AEPs are observed regenerating AT2 cells. d, Krt5+ cells are distinct from AEPs and never bear the AEP lineage mark. Red arrow, Krt5+ cell. e, AEP-lineage cells do not express Krt5 or Sox2 protein at baseline, in contrast to previously reported lineages7,8. Arrows represent probable AEPs by morphology. f, Krt5+ cells predominate in zone 4 regions, where AEPs are not present. g, Quantification demonstrating that Krt5+ cells are never marked with the AEP lineage mark. h, AT2 populations expand markedly after influenza injury, except in zone 4. i, Krt5+ cells rarely express Sftpc in zone 4 regions. jl, One month after influenza injury, AEPs give rise to a small number of Hopx+ AT1 cells, predominantly in zone 2 of mild injury. Yellow arrow, AEP-labelled cells; white arrow, unlabelled cells. Zone 3 (l) has very few AEP-derived Hopx+ cells, which may be due to a lag in AT1 regeneration from AEPs in this more severely affected region. Data shown represent n = 6 (ag, i) or 10 (h, j, k) independent mice across three individual experiments. Statistics are representative of all biological replicates. All data are shown as centred on mean with bars indicating standard deviation. **P < 0.01 and ***P < 0.001, by ANOVA with preplanned pairwise comparisons and adjustment for multiple comparison testing. Scale bars: a, 200 μm; b–d, j–l, 50 μm. Source data

Extended Data Figure 5 Wnt signalling in the alveolar epithelium is largely stable after influenza infection, and AEP lineage labelling is not affected by tamoxifen perdurance.

a, FACS gating strategy used for all post-influenza FACS experiments in Fig. 1, Extended Data Fig. 2 and b, c. SSC-A, side-scatter area, SSC-H, side-scatter height, FSC-H, forward-scatter height. b, c, FACS analysis demonstrates that Axin2tdT intensity is mildly decreased in the epithelium at 7 and 14 days after influenza infection. d, In regions of milder lung injury, most lineage-labelled AT2 cells are eYFP+ and tdTomato, which suggests that these cells are the progeny of AEPs. e, In zone 3, we detect a mix of eYFP+tdTomato+ AEPs (red arrowheads) and eYFP+tdTomato AEP progeny (yellow arrowheads) among the AT2 cell population. f, Experimental design of lineage tracing experiment in gi, with a longer incubation time after tamoxifen treatment than in the experiments that generated the data presented in ae, and Fig. 1 and Extended Data Figs 4, 6. g, h, Confocal imaging demonstrating lineage labelling of AT2 cells with the AEP lineage mark 28 days after influenza-mediated injury. White arrows, unlabelled AT2 cells; yellow arrows, AEP-labelled cells. i, Quantification of lineage-labelled AT2 cells in multiple regions of lung injury. Representative seven-day lineage data is reproduced from Fig. 1 for comparison. Data shown represent n = 4 (ac) or 5 (di) independent mice across two different experiments. Statistics are representative of all biological replicates. All data were analysed with ANOVA followed by preplanned pairwise comparisons and adjustment for multiple comparison testing, and are shown centred on mean with bars indicating standard deviation. **P < 0.01. Scale bars, 50 μm. Source data

Extended Data Figure 6 Transcriptome analysis of AEPs versus AT2 cells, and activation of cell-cycle genes in AEPs after influenza injury.

a,Volcano plot of 14,618 genes tested using a linear model in the R package limma, showing the distinct differences in gene expression in AEPs and AT2 cells. Notable lung-progenitor developmental signalling and transcription factors are indicated. b, GO analysis of the top 500 most-differentially expressed genes, showing the enrichment of categories related to lung development and morphogenesis in AEPs. c, Heat maps of two of the AEP-enriched GO categories. Important regulators of lung-progenitor-cell biology are indicated. d, qPCR confirms upregulation of a subset of important regulators of lung progenitor biology in AEPs. e, AT2 and AEP open chromatin is found near distinct sets of genes involved in the cell cycle. f, Schematic of analysis of changes in expression of AEP-primed genes after influenza infection. g, A subset of primed cell-cycle regulators in AEPs show expression changes after influenza infection. qPCR data are from n = 4 mice from two separate infections. All data are shown as centred on mean with bars indicating standard deviation. Statistics are representative of all biological replicates. *P < 0.05 and **P < 0.01 by two-tailed t-test. Source data

Extended Data Figure 7 ATAC-seq reveals distinct differences in open chromatin architecture in AEPs versus AT2 cells.

a, ATAC-seq peaks in both AT2 cells and AEPs are similar to previously described34 mouse lung genome-wide DNase hypersensitivity profiling. b, AT2 and AEP ATAC peaks are distributed in a similar fashion, predominantly within intergenic regions and introns. c, GO enrichment analysis of the nearest neighbour genes in the vicinity of AT2 peaks, AEP peaks and peaks common to both AEPs and AT2 cells shows that common peaks are enriched for general cellular housekeeping roles, whereas AT2 open chromatin is enriched near genes associated with exocytosis and cell differentiation. By contrast, AEP peaks are enriched near genes associated with lung development processes. d, e, Examination of the genes associated with open chromatin in AEPs reveals a strong enrichment for transcription factors associated with lung endoderm progenitor cells, including members of Klf, Six, Sox, Nkx2 and Elf/Ets families. By contrast, AT2 cell open chromatin is associated with a unique set of transcriptional regulators that includes members of the NfI and Cebp families, which are known to regulate AT2 cell surfactant genes. For details of ATAC analysis, see Methods. Source data

Extended Data Figure 8 The combination of HT2-280 and TM4SF1 antibodies are capable of identifying AEPs in human lung.

a, Top panels show isotype and active antibody gates for sheep anti-mouse Tm4sf1 FACS. The bottom panels show that the Tm4sf1 antibody detects approximately 20% of SftpccreERT2eYFP labelled AT2 cells. b, Isotype and active antibody gates for human HT2-280 (AT2 marker) antibody and TM4SF1 antibody. c, d, An example of the FACS gating strategy used to generate the data shown in Fig. 3. e, f, Selection for HT2-280 strongly enriches for human AT2 cells. g, h, The majority of isolated HT2-280 cells express SFTPC protein by cytospin. i, j, Human AEPs in organoid culture do not express KRT5 or SOX2 protein at detectable levels. Each FACS panel shown in af shows gates from cells of one individual mouse or patient and is representative of n= 6 independent mice across two individual experiments or n = 4 human patients. Isotype staining was performed three times to confirm specificity. Statistics are representative of all biological replicates. Statistics in h are calculated with two-tailed t-test, displayed as mean with bars showing standard deviation. Scale bars: g, 25 μm; i, j, 50 μm (i, j). Source data

Extended Data Figure 9 Mouse AEPs generate more alveolar organoids compared to AT2 cells, and cells in these organoids are restricted from AT1 cell differentiation by Wnt signalling.

a, Schematic of mouse alveolar organoid culture method. bm, Sftpc+ mouse AT2 cells (bd, hj) and mouse AEPs (eg, km) were isolated from the indicated mouse lines and cultured in alveolar organoid assays. AT2 cells (b) and AEPs (e) both form alveolar organoids. AEPs generate more numerous and larger organoids than do AT2 cells. Activation of Wnt signalling using CHIR99021 does not increase the organoid-forming efficiency of either AT2 cells (c) or AEPs (f) but does increase the number of Sftpc+ cells in treated organoids (i, l, o). Inhibition of Wnt signalling using XAV939 increases the number and size of alveolar organoids (d, g, n, q), decreases the number of Sftpc+ AT2 cells and increases the number of Aqp5+ AT1 cells (j, m, p). For tests of all parameters, AEPs exhibited a more marked response to Wnt modulation than did AT2 cells. Data shown represent n = 12 wells from n = 4 individual mice in each group, across 3 individual experiments. Quantitative counting shown for cell differentiation (o, p) represents counting of n > 400 organoids from n = 4 mice. All data were analysed with ANOVA followed by preplanned pairwise comparisons and adjustment for multiple comparison testing, and are shown centred on mean with bars indicating standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Statistics are representative of all biological replicates. Scale bars: 50 μm. Source data

Extended Data Figure 10 Combination of ATAC-seq and RNA-seq emphasizes the Wnt- and FGF-responsive nature of AEPs and identifies several novel AEP-enriched direct Wnt target genes.

a, Schematic of human RNA-seq experiments. b, GO term analysis of the top 300 human AEP-enriched genes shows enrichment of several categories associated with lung progenitor cell function, similar to observations made of mouse AEPs. c, Evaluation of chromatin accessibility in the mouse genome near common AEP-enriched genes demonstrates a significant overrepresentation of Tcf binding sites, particularly in putative regulatory regions 5 kb immediately upstream of the transcriptional start site. For details of enrichment analysis, see Methods. d, Schematic of areas of AEP-enriched open chromatin near selected AEP-enriched genes. Peak height represents coverage of the indicated genomic region in the ATAC library, and the number indicates the fold enrichment in the indicated peak. e, Chromatin immunopreciptiation qPCR on AEP versus AT2 chromatin demonstrates Ctnnb1 antibody binding at the differentially accessible genomic regions near Etv4, Sftpa, Lamp3 and Gpr116 in AEP cells, indicating that these genes are direct Wnt targets. Data are shown as mean with individual data points showing summary data from two independent chromatin immunopreciptiation experiments with multiple technical replicates. fj, Fgfr2 activation in mouse AEPs drives increased proliferation and the formation of larger organoids; quantification shown in j. See Fig. 4 for additional data. k, RNAscope showing enriched expression of Fgfr2 (red) in lineage-labelled AEPs. lq, Similar to treatment with Fgf7, Fgf10 treatment drives increased colony-forming efficiency in both mouse AEPs (lp) and human AEPs (q). Data shown in fj, lq represent a minimum of n = 12 wells across two individual experiments. Statistics are representative of all biological replicates. Data were analysed with ANOVA followed by preplanned pairwise comparisons and adjustment for multiple comparison testing, and are shown centred on mean with bars indicating standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Source data

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Supplementary Table 1

Genes differentially expressed in mouse AEPs compared to mouse AT2 cells (XLSX 182 kb)

Supplementary Table 2

Genes differentially expressed in human AEPs compared to other human AT2 cells. (XLSX 97 kb)

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Zacharias, W., Frank, D., Zepp, J. et al. Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor. Nature 555, 251–255 (2018). https://doi.org/10.1038/nature25786

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