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Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury


Broadly, tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ- and injury-specific1,2,3,4. Current models in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers5,6,7,8. By contrast, here we define the regenerative role of previously uncharacterized, rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Quiescent LNEPs activate a ΔNp63 (a p63 splice variant) and cytokeratin 5 remodelling program after influenza or bleomycin injury in mice. Activated cells proliferate and migrate widely to occupy heavily injured areas depleted of mature lineages, at which point they differentiate towards mature epithelium. Lineage tracing revealed scant contribution of pre-existing mature epithelial cells in such repair, whereas orthotopic transplantation of LNEPs, isolated by a definitive surface profile identified through single-cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population. LNEPs require Notch signalling to activate the ΔNp63 and cytokeratin 5 program, and subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signalling after injury led to parenchymal ‘micro-honeycombing’ (alveolar cysts), indicative of failed regeneration. Lungs from patients with fibrosis show analogous honeycomb cysts with evidence of hyperactive Notch signalling. Our findings indicate that distinct stem/progenitor cell pools repopulate injured tissue depending on the extent of the injury, and the outcomes of regeneration or fibrosis may depend in part on the dynamics of LNEP Notch signalling.

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Figure 1: Injury-induced Krt5+ cells are derived from a lineage-negative precursor.
Figure 2: Isolation and transplantation of a lineage-negative distal epithelial population.
Figure 3: Activation and Krt5 expression by lineage-negative progenitors is Notch-dependent.
Figure 4: Persistent Notch activity promotes cystic honeycombing in both mouse and human.

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Gene Expression Omnibus

Data deposits

The FPKM files from single-cell RNA-seq experiments have been deposited in Gene Expression Omnibus (GEO) under accession GSE61300.


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This work was supported by National Institutes of Health (NIH) grants RO1 HL44712 and UO1 HL111054 and a sponsored research agreement with Daiichi Pharmaceuticals. A.E.V. is supported by F32 HL117600-01. The authors thank T. Kim for assistance with animal work and K. Corbin for assistance with Imaris software. Mouse line art was created by A. van de Wiel. We also thank P. Wolters at the UCSF Interstitial Lung Disease Blood and Tissue Repository for procuring diseased lung tissues. We thank the Nina Ireland Program for Lung Health that supported the tracheal/lung transplant experiments.

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Authors and Affiliations



A.E.V. and H.A.C. designed the study, analysed the data, and wrote the manuscript. A.E.V. performed lineage tracing, flow cytometry purification, and characterization of lung cells; J.E.G. titred PR8 virus and initiated all infections; A.N.B. isolated lung cell suspensions, assisted with flow cytometry, and designed and performed most of the immunostaining; Y.X. assisted with biochemistry, RNA analysis, and immunostaining; K.T. and V.T. managed the mouse genotyping and performed in vivo mouse experiments; V.T. isolated lung cells and designed quantification methods; F.C.L. and M.R.L. performed lung transplantations; M.M. procured and screened human lungs; D.G.B. and B.T. synthesized libraries and provided initial data analysis for RNA-seq experiments J.R.R. provided key reagents and assisted with study design.

Corresponding authors

Correspondence to Andrew E. Vaughan or Harold A. Chapman.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Characterization of influenza-induced Krt5+ cells.

ac, Alveolar (a, b) and airway (c) Krt5+ cells strongly express β4 after influenza injury. d, FACS plot of epithelial (EpCAM+) cells from tamoxifen-treated Krt5-CreERT2/tdTomato mice at day 15 after influenza, demonstrating β4 expression in nearly all traced (tdTomato+) cells. e, f, Most Krt5+ cells co-express ΔNp63 (e) and Krt14 (f). g, h, Expanded Krt5+ cells are invariably associated with abundant CD45+ inflammatory cells (g) and few if any remaining normal E-cadherin+ epithelial cells other than the Krt5+ cells themselves (h). i, Krt5+ cells are unlabelled in SPC-CreERT2/mTmG mice. Inset in i demonstrates appropriate labelling of type II cells in an uninjured region of the same lung. j, k, Krt5+ cells are not fluorescent after trachea transplantation from tdTomato donor. Basal cells in transplanted section of trachea retained fluorescence (j, inset in k). Scale bars, 100 μm (a) and 20 μm (b, c, ek).

Extended Data Figure 2 Influenza-induced Krt5+ cells arise in both airways and alveoli and migrate across, around and through airway and parenchymal tissue.

a, b, Krt5+ cells are detected in alveoli as early as day 5 and are found in larger clusters over time. c, d, Krt5+ cells similarly arise in airways in greater abundance with time. e, Distinct alveolar and airway expansion is apparent 11 days after infection. f, Freeze-frames of live imaging from a Krt5-CreERT2/tdTomato mouse 11 days after influenza, in which tdTomato+ cells migrate from their original location (white box) outward. See Supplementary Video 1. g, Freeze-frames from a small airway in the same mouse; arrow denotes a single cell crossing the basement membrane. See Supplementary Video 2. Scale bars, 20 μm (a, b, g) and 100 μm (c, d, f).

Extended Data Figure 3 Characterization of bleomycin-induced Krt5+ cells.

ac, β4+ Krt5+ cells also arise after bleomycin injury and express ΔNp63 (b, c). d, Western blotting demonstrating more pronounced and reproducible Krt5 induction after influenza injury at day 11 than after bleomycin injury at day 17. Each lane was loaded with whole-lung lysate from a single mouse; average percentage lung area corresponding to a band in influenza-injured mice is 3.6 ± 0.5% (n = 13 mice quantified, see Fig. 3g as an example). e, Lineage tracing of bleomycin-injured Krt5-CreERT2 mice reveal traced (tdTomato+) type II cells expressing SPC and cells morphologically resembling type I cells. In total, 31% of Krt5-CreERT2 traced cells express SPC by day 50 after bleomycin (n = 3 mice, 264 Krt5-CreERT2-labelled cells counted). Scale bars, 100 μm (a) and 20 μm (b, c, e). Full western blot scan in d is available as Supplementary Fig. 1.

Extended Data Figure 4 Krt5+ cells do not arise from CC10-expressing progenitors but rather upregulate CC10 during expansion.

a, Krt5+ cells express detectable levels of CC10 (top) compared to isotype control (bottom) in alveolar clusters (a). b, Representative image of CC10-CreERT2 lineage trace in which waiting only 7 days after tamoxifen administration before influenza injury results in significant labelling of Krt5+ cells (quantified in Fig. 1d). c, Strong CC10 expression in Krt5-CreERT2-traced (tdTomato+) cells by day 22 after influenza. For comparison, see single channel images (c, right and bottom) of the same region. Scale bars, 20 μm.

Extended Data Figure 5 Heterogeneity of the LNEP-containing CC10 β4+ population.

a, Rare Krt5-CreERT2-traced (tdTomato+) cells were observed in uninjured distal lung airways that lacked Krt5 staining compared to trachea basal cells (inset) in the same section. All distal tdTomato+ cells express ΔNp63 but most ΔNp63+ cells are untraced (see Fig. 2c). b, Cytospins of sorted CC10 β4+ cells reveal the presence of abundant multiciliated cells (green, acetylated tubulin+) and a small fraction of ΔNp63+ cells (red). c, Quantitative reverse transcriptase PCR (qRT–PCR) analysis of mature lineage genes and genes of interest in all populations. n = 3 biological replicates; data are mean ± s.d. d, Principal component analysis plot of cells sequenced in Fig. 2b, demonstrating that p63+ cells in the CC10 β4+ population (outlined, asterisk) cluster with multi-ciliated cells. e, CD200 is not expressed by FoxJ1-CreERT2-labelled multi-ciliated cells, highlighting its use in excluding such cells. f, Cytospin of Foxj1-CreERT2-labelled β4+ cells demonstrating reliable selection for multi-ciliated cells (198 cells quantified). g, Gating on CD14 expression within the EpCAM+ β4+ CD200+ population excludes CC10-expressing club cells. Scale bars, 20 μm.

Extended Data Figure 6 Orthotopic transplantation of LNEPs reveals their multipotency and differentiation appropriate to the local microenvironment.

a, Several distinct areas of LNEP engraftment (red) reflect differentiation in response to location. Left dashed box demonstrates SPC expression in engrafted cells with nearby endogenous SPC-expressing cells (white); far right dashed box demonstrates Krt5 expression in engrafted cells and nearby endogenous Krt5-expressing cells (green). b, c, Cells in regions of SPC+ differentiation (b) lack Hes1 expression (right), whereas those in areas of Krt5+ differentiation (c) strongly express Hes1 (right). d, Distinct areas of LNEP engraftment demonstrate an inverse relationship between SPC expression (left) and Hes1 expression (right) in probable single clones. e, Examination of transplanted cells 5 days after engraftment demonstrate abundant Edu incorporation (see Methods) indicative of proliferation. At this time point cells can be identified co-expressing β4 and SPC (right, circled). f, g, Krt5+ cells and CC10+ cells were often found clustered in single regions of engraftment. h, Many engrafted cells in Fig. 2e are also SPC positive. i, β4 type II cells engraft in small clusters and only express SPC. j, k, CC10+ cells engraft but do not express SPC, CC10 or Krt5. l, Multi-ciliated cells engraft but only persist as isolated single cells, losing acetylated tubulin expression. Scale bars, 100 μm (a) and 20 μm (bl).

Extended Data Figure 7 Transplantation of β4+ CD14+ CD200+ and Krt5-CreERT2-traced cells recapitulates multipotency of the heterogenous CC10 β4+ population.

a, Single channels images from Fig. 2h demonstrate Krt5 expression in transplanted β4+ CD14+ CD200+ cells. b, c, Transplanted β4+ CD14+ CD200+ can also differentiate towards type II cells (b) and club cells (c). d, e, Transplantation of rare Krt5-CreERT2-traced cells from uninjured mice resulting in donor-derived Krt5+ cell expansion indistinguishable from endogenous expansion. Images in d and e are representative images from four attempted transplants, two of which exhibited engraftment in two or four individual lobes. Scale bars, 20 μm.

Extended Data Figure 8 Notch activity in normal and injured lung.

a, Uninjured Notch reporter mice (Cp-eGFP) show dim GFP in small airways and no detectable GFP in alveoli. b, Krt5+ cells arising in distal airways express GFP in Notch reporter mice 7 days after influenza infection. c, d, Some Krt5+ cells persist within Krt5-CreERT2-labelled (tdTomato+) cysts (d) long-term (day 88) after influenza injury, and many traced cells express CC10 (c). e, Cysts rarely contain SPC+ type II cells (arrows). f, g, Hes1 expression is maintained in Krt5-CreERT2-traced (GFP+) cyst cells 98 days after influenza (f) but is absent in normal alveolar parenchyma from the same mice (g). h, Representative images of Krt5+ cell expansion in vehicle- (left) or DAPT- (right) treated mice at day 11 after influenza, quantified in Fig. 3g. Scale bars, 20 μm (ag) and 100 μm (h).

Extended Data Figure 9 IPF and scleroderma lungs both contain HES1+ honeycomb cysts, but scleroderma lungs also possess SPC and KRT5 co-expressing cells.

Normal human lungs contain putative LNEPs and lack HES1 in alveoli. ad, Honeycomb cysts in several IPF lungs; many KRT5+ cells as well as surrounding cystic epithelium demonstrate strong nuclear HES1 signal. e, Region of scleroderma honeycombing similar to IPF lung. f, Scleroderma subpleural alveolar region with type II cell hyperplasia demonstrating cells co-expressing SPC and KRT5. g, h, Cystic epithelium in scleroderma lungs expresses HES1 as in IPF. i, KRT5 ΔNp63+ cells (white outlines) distinct from KRT5+ ΔNp63+ basal cells (red outlines) are present in distal airways. j, k, HES1 staining is apparent in small airways of normal lung (j) but very low in alveolar parenchyma (k). All images are from patient samples in addition to those shown in Fig. 4. Scale bars, 20 μm (ad, gk) and 100 μm (e, f).

Extended Data Figure 10 Hierarchical cellular responses to injury severity and Notch-regulated LNEP dynamics.

a, Distinct epithelial cell types contribute to regeneration depending on the severity of parenchymal injury. Examples of each are referenced. b, Notch signalling regulates the activation, expansion and differentiation of LNEPs. Notch is required for activation and maintenance of LNEPs. Alveolar differentiation requires subsequent loss of Notch activity, whereas persistent Notch results in either airway differentiation or abnormal cystic honeycombing.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion with additional references, Supplementary Figure 1 and Supplementary Tables 1 and 2. (PDF 4085 kb)

Supplementary Data 1: Genes enriched in putative LNEP population.

Excel file generated with Singular via ANOVA analysis of differential expression. Genes are sorted by fold change and column filters set for FPKM > 1, > 2 fold higher expression in LNEPs, ANOVA p-value ≤0.05. (XLSX 1914 kb)

Supplementary Data 2: Genes enriched in putative multiciliated cells.

Excel file generated with Singular via ANOVA analysis of differential expression. Genes are sorted by fold change and column filters set for FPKM > 1, > 2 fold higher expression in multiciliated cells, ANOVA p-value ≤0.05. (XLSX 2336 kb)

Migration of Krt5+ cells after injury

Krt5+ cells discriminated by tdTomato expression migrate widely over damaged lung parenchyma in a lung slice culture from a Krt5-CreERT2 / tdTomato mouse at day 11 post-influenza. (MOV 1061 kb)

Migration of Krt5+ cells after injury

Higher power view of an airway in Video 1 in which a single tdTomato+ cells migrates around an airway and appears to cross the basement membrane into adjacent parenchymal space. Video frames may be advanced manually in order to view in more detail. (MOV 1867 kb)

Alveolar Krt5+ cells extend into neighboring alveoli upon injury

tdTomato+ cells from Krt5-CreERT2 / tdTomato mouse at day 5 post-influenza sends extensions into neighboring alveoli. (AVI 378 kb)

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Vaughan, A., Brumwell, A., Xi, Y. et al. Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury. Nature 517, 621–625 (2015).

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