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ABSTRACT Lung diseases such as chronic obstructive pulmonary disease1 and pulmonary fibrosis2 involve the progressive and inexorable destruction of oxygen exchange surfaces and airways, and

have emerged as a leading cause of death worldwide. Mitigating therapies, aside from impractical organ transplantation, remain limited and the possibility of regenerative medicine has lacked

empirical support. However, it is clinically known that patients who survive sudden, massive loss of lung tissue from necrotizing pneumonia3,4 or acute respiratory distress syndrome5,6

often recover full pulmonary function within six months. Correspondingly, we recently demonstrated lung regeneration in mice following H1N1 influenza virus infection, and linked distal

airway stem cells expressing Trp63 (p63) and keratin 5, called DASCp63/Krt5, to this process7. Here we show that pre-existing, intrinsically committed DASCp63/Krt5 undergo a proliferative

expansion in response to influenza-induced lung damage, and assemble into nascent alveoli at sites of interstitial lung inflammation. We also show that the selective ablation of DASCp63/Krt5

_in vivo_ prevents this regeneration, leading to pre-fibrotic lesions and deficient oxygen exchange. Finally, we demonstrate that single DASCp63/Krt5-derived pedigrees differentiate to type

I and type II pneumocytes as well as bronchiolar secretory cells following transplantation to infected lung and also minimize the structural consequences of endogenous stem cell loss on

this process. The ability to propagate these cells in culture while maintaining their intrinsic lineage commitment suggests their potential in stem cell-based therapies for acute and chronic

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Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS CLONED AIRWAY BASAL PROGENITOR CELLS TO REPAIR FIBROTIC LUNG THROUGH RE-EPITHELIALIZATION Article Open access 03 February 2025

HUMAN RESPIRATORY AIRWAY PROGENITORS DERIVED FROM PLURIPOTENT CELLS GENERATE ALVEOLAR EPITHELIAL CELLS AND MODEL PULMONARY FIBROSIS Article 24 February 2025 SINGLE-CELL DIVISION TRACING AND

TRANSCRIPTOMICS REVEAL CELL TYPES AND DIFFERENTIATION PATHS IN THE REGENERATING LUNG Article Open access 12 March 2024 ACCESSION CODES PRIMARY ACCESSIONS GENE EXPRESSION OMNIBUS * GSE60849

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CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by grants from the Joint Council Office of the Agency for Science Technology Research Agency (ASTAR),

Singapore (W.X., F.M.), Defense Advanced Research Projects Agency (DARPA, N66001-09-1-2121 to F.M.), the Johnson & Johnson ASTAR Joint Program (W.X., F.M.) and support from Connecticut

Innovations (W.X., F.M.). We thank M. LaLande, B. Lane and H. Hui Ng for support, G. Wright, B. Tennent, B. Knowles and T. McLaughlin for comments on the manuscript, J. Hammer for artwork,

P. Kraus for blastocyst injections, and H. Ahmad and K. L. Goh for technical assistance. We thank H. Green for advice and support. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Genome

Institute of Singapore, A-STAR, 138672 Singapore, Wei Zuo, Ting Zhang, Daniel Zheng'An Wu, Shou Ping Guan, Audrey-Ann Liew, Siew Joo Lim, Wa Xian & Frank McKeon * The Jackson

Laboratory for Genomic Medicine, Farmington, 06032, Connecticut, USA Yusuke Yamamoto, Xia Wang, Wa Xian & Frank McKeon * Advanced Cell Technologies, Marlborough, 01752, Massachusetts,

USA Matthew Vincent * The Jackson Laboratory, Bar Harbor, 04609, Maine, USA Mark Lessard * Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, 02115,

Massachusetts, USA Christopher P. Crum & Wa Xian * Department of Medicine, National University Health System, 119228 Singapore, Wa Xian & Frank McKeon * Department of Genetics and

Developmental Biology, University of Connecticut Health Center, Farmington, 06030, Connecticut, USA Wa Xian Authors * Wei Zuo View author publications You can also search for this author

inPubMed Google Scholar * Ting Zhang View author publications You can also search for this author inPubMed Google Scholar * Daniel Zheng'An Wu View author publications You can also

search for this author inPubMed Google Scholar * Shou Ping Guan View author publications You can also search for this author inPubMed Google Scholar * Audrey-Ann Liew View author

publications You can also search for this author inPubMed Google Scholar * Yusuke Yamamoto View author publications You can also search for this author inPubMed Google Scholar * Xia Wang

View author publications You can also search for this author inPubMed Google Scholar * Siew Joo Lim View author publications You can also search for this author inPubMed Google Scholar *

Matthew Vincent View author publications You can also search for this author inPubMed Google Scholar * Mark Lessard View author publications You can also search for this author inPubMed 

Google Scholar * Christopher P. Crum View author publications You can also search for this author inPubMed Google Scholar * Wa Xian View author publications You can also search for this

author inPubMed Google Scholar * Frank McKeon View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Experiments were performed by W.Z., D.Z.W.,

S.P.G. and A.-A.L. Experimental design and conception were done by W.Z., M.V., C.P.C., W.X. and F.M.; T.Z., W.Z., X.W., S.J.L. and Y.Y. performed microarrays and computational analysis, and

provided methodological advice. M.L. and W.Z. performed the serial reconstructions of infected lung. CORRESPONDING AUTHORS Correspondence to Wa Xian or Frank McKeon. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing financial interests. ADDITIONAL INFORMATION Datasets generated for this study have been submitted to the National Center for

Biotechnology Information Gene Expression Omnibus (GEO) database under superseries GSE60849. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1 LINEAGE TRACING OF KRT5+ CELLS. A, Left,

immunofluorescence images of sections of 15 dpi lung with staining patterns of antibodies to pan-leukocyte marker CD45 and the type I pneumocyte marker Pdpn with DNA counterstained with

DAPI. Right, immunofluorescence images of pan-leukocyte marker CD45 and the type II pneumocyte marker SPC. Scale bar, 150 μm. B, X-gal staining (blue) to reveal lacZ-dependent

β-galactosidase activity in whole lungs after 15 and 40 days post infection following long time gaps between induction of lacZ labelling by tamoxifen and influenza infection-induced lung

damage. The similarity of this long gap labelling and the short gap labelling presented in Fig. 1 argues against prolonged actions of tamoxifen in these lineage-labelling protocols.

Tamoxifen is given at indicated times before infection and no-tamoxifen control is included. C, Immunofluorescence images of colonies of DASCs derived from tamoxifen-treated ROSA26-lsl-lacZ;

Krt5-CreERT2 mice stained with antibodies to keratin 5 (Krt5) or Krt5 and _E. coli_-specific β-galactosidase. D, Histological section of lung at 15 dpi stained with _E. coli_-specific

β-galactosidase antibody and markers of secretory cells (CC10+) and expanded stem cells (Krt5+). Scale bar, 50 μm. E, Whole-mount image of X-gal developed, uninfected lung from

ROSA26-lsl-lacZ; Krt5-CreERT2 which received tamoxifen treatments at −69, −66 and −63 days before dissection. F, Histological section of 60 dpi lung stained with _E. coli_-specific

β-galactosidase antibody and markers of type I pneumocytes (Pdpn+) and type II pneumocytes (SPC+). EXTENDED DATA FIGURE 2 CONDITIONAL DASCP63/KRT5 ABLATION MOUSE MODEL. A, Schematic of

_Krt6a_ locus, the targeting vector constructed to introduce the human diphtheria toxin receptor (DTR). B, The structure of the modified _Krt6a_ locus in embryonic stem cells screened by

Southern blot. C, Co-expression of Krt6 and DTR in Krt5+ pods in 15 dpi lung. D, Histogram showing resistance of wild-type, 12 dpi DASCp63/Krt5 to diphtheria toxin (DTox) and the sensitivity

of DASCp63/Krt5/DTR to diphtheria toxin. _n_ = 3 mice per group. Error bars, s.e.m. EXTENDED DATA FIGURE 3 PERSISTENT DAMAGE IN DASCP63/KRT5-ABLATED LUNGS. A, Gene set enrichment analysis

(GSEA) showing the overrepresentation of normal alveolar signature gene sets in WT rather than Krt6–DTR mouse lungs (whole lobes). For normal alveolar signature build up, laser capture

microdissection of frozen sections was used to dissect normal alveoli region from 0 dpi lung and damaged interstitial infiltrated region from 15 dpi lung for microarray analysis.

Differentially expressed genes (fold change > 5, _P_ < 0.01) were used to develop normal alveolar gene expression signatures. B, Top panel, histological analysis of lung densities

using anti-Pdpn antibodies (red) and anti-CD45 (green) to reveal type I pneumocytes and leukocyte infiltration, respectively. Left, wild-type mice showing apparently normal lung region

adjacent to interstitial density having Pdpn+ network but lacking CD45+ infiltrates. Right, Krt6–DTR lung showing apparently normal region adjacent to zone of damaged interstitial lung

lacking Pdpn+ network but having CD45+ infiltrates. Bottom panel, H&E staining of the same histological region. Scale bar, 100 μm. EXTENDED DATA FIGURE 4 NETWORKS OF TYPE I PNEUMOCYTES

IN 30 DPI MOUSE LUNG. A, Histological analysis of lung densities using anti-Pdpn antibodies (red) and anti-SPC (green) to reveal type I and type II pneumocytes respectively. Left, wild-type

mice showing apparently normal lung region adjacent to interstitial density having Pdpn+ network but lacking SPC+ cells. Right, Krt6–DTR lung showing normal region adjacent to zone of

damaged interstitial lung lacking both pneumocytes. Scale bar, 100 μm. B, Top panel, histological analysis of wild-type lung densities using anti-Pdpn antibodies (red) and anti-Aqp5 (green)

type I pneumocyte markers showing the interstitial density having Pdpn/Aqp5 double-positive network. Bottom panel, wild-type mice show apparently normal lung region adjacent to interstitial

density having Pdpn+ network but the density lack expression of another type I pneumocyte marker, Hopx. Scale bar, 100 μm. EXTENDED DATA FIGURE 5 FAILURE OF REGENERATION IN

DASCP63/KRT5-ABLATED LUNGS. A, Histological section through 30 dpi DASC-ablated lung (Krt6–DTR +DTox) showing normal region (Pdpn+) adjacent to interstitial density positive for α-SMA and

weakly positive for Masson’s trichrome (MT) staining for fibrosis. Scale bar, 100 μm. B, Histological section through 30 dpi control lung showing normal region (Pdpn+) and interstitial

density (Pdpn−) which are both negative for α-SMA. C, Expression heat map of selected, differentially expressed genes (_P_ < 0.05) comparing wild-type mouse lungs with DASC-ablated mouse

lungs at 30 dpi. Scale bar, 100 μm. EXTENDED DATA FIGURE 6 CLONING AND _IN VITRO_ DIFFERENTIATION OF TBSCP63/KRT5 AND DASCP63/KRT5. A, Histogram of cloning efficiency of TBSCs and DASCs on

irradiated 3T3-J2 cells per 1 million tracheal or distal airway cells derived from respective tissues of adult mice. Tissues derived from 3 mice. Error bars, s.e.m. B, Immunofluorescence

images of sections of TBSC and DASC air–liquid interface cultures using an antibody to the type I pneumocyte marker Pdpn (green). Sections were counterstained with DAPI (blue). EXTENDED DATA

FIGURE 7 TRANSPLANTATION OF DASCLACZ. A, Immunofluorescence characterization of DASCs isolated from Krt5-CreERT2;ROSA26-lsl-lacZ mice following Cre activation with 4OH-tamoxifen. From left,

colony stained with antibodies to p63 (green) and Krt5 (red), p63 (green) and _E. coli_ β-galactosidase (red), Krt5 (red) and CC10 (green), and Krt5 (red) and SPC (green). B, Whole mount

image of lung 90 days after infection without stem cell transplantation. C, Left, bright field/immunofluorescence image of section of lung at 90 dpi following transplantation of DASClacZ

stained with antibodies to β-galactosidase (red). Right panels, immunofluorescence images of co-staining of transplanted DASClacZ with antibodies to Pdpn, SPC, or CC10 at high magnification.

EXTENDED DATA FIGURE 8 PERSISTENT PROLIFERATION OF TRANSPLANTED DASC. Co-staining of antibodies to GFP (green) with the cell proliferation marker Ki67 (red) in sections of lung transplanted

with DASCGFP at 12 dpi lung (7 days post transplantation) and 60 dpi lung (55 days post transplantation). Top left, immunofluorescence image of lung following transplantation of DASCGFP (7 

days post-transplantation;12 dpi) stained with anti-GFP (green) and the cell cycle marker Ki67 (red, in nucleus). Top right, bronchiole co-stained with antibodies to GFP and Ki67 from 7 days

post-transplantation lung. Bottom, staining of interstitial lung transplanted 55 days prior with DASCGFP with antibodies to GFP and Ki67. Arrows indicate cells co-expressing GFP and Ki67.

EXTENDED DATA FIGURE 9 STEM CELL TRANSPLANTATION REDUCES INTERSTITIAL DENSITIES IN DASCP63/KRT5-ABLATED LUNGS. A, Histological sections through entire lobe of Krt6–DTR mice with (left) and

without (right) diphtheria toxin treatment forty days post-influenza infection. B, Histogram of morphometric quantification of lung densities following 40 day influenza virus infection of

Krt6–DTR mice without diphtheria toxin (−DTox, mouse number _n_ = 3), with diphtheria toxin (+DTox, _n_ = 4), or with diphtheria toxin and transplanted DASCs (+DTox+DASC, _n_ = 4). Error

bars indicate s.e.m. and # indicates _P_ value = 0.029 by Wilcoxon rank-sum test. SUPPLEMENTARY INFORMATION RECONSTRUCTION OF 3-D PATTERN OF KRT5 STAINING WITH RESPECT TO BRONCHIOLES (BLUE)

IN PORTION OF 15DPI INFECTED LUNG FROM SERIAL SECTIONS. Reconstruction of 3-D pattern of Krt5 staining with respect to bronchioles (blue) in portion of 15dpi infected lung from serial

sections. (MP4 27357 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 RIGHTS AND PERMISSIONS Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Zuo, W., Zhang, T., Wu, D. _et al._ p63+Krt5+ distal airway stem cells are essential for lung regeneration. _Nature_ 517, 616–620 (2015).

https://doi.org/10.1038/nature13903 Download citation * Received: 30 March 2014 * Accepted: 30 September 2014 * Published: 12 November 2014 * Issue Date: 29 January 2015 * DOI:

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