p63(+)Krt5(+) distal airway stem cells are essential for lung regeneration

Abstract

Lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis 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 pneumonia or acute respiratory distress syndrome 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 DASC(p63/Krt5), to this process. Here we show that pre-existing, intrinsically committed DASC(p63/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 DASC(p63/Krt5) in vivo prevents this regeneration, leading to pre-fibrotic lesions and deficient oxygen exchange. Finally, we demonstrate that single DASC(p63/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 lung diseases.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Lineage tracing of Krt5+ cells following viral infection.

a, Left, mouse lung before and after viral infection. Right, immunofluorescence images of infected lung of anti- Krt5 (red), anti-Pdpn (green) with DNA counterstain (DAPI, blue). Scale bar, 1 mm. Insets, high magnification of indicated regions. n = 10 mice. Scale bars, 100 μm. b, Three-dimensional reconstruction of anti-Krt5 (red) from serial sections of infected lung (bronchioles, blue). Grid, 100 × 100 μm. c, Schematic of lineage tracing experiment following tamoxifen treatment to reveal lacZ expression 0, 9, 15 and 60 days post infection (n = 3 mice for each time point and control). At 0 dpi, rare clusters of p63+ cells in bronchioles express E. coli β-galactosidase (left top, arrows). Scale bar, 20 μm. Colony of Krt5+ DASC co-expressing β-galactosidase at 0 dpi (left bottom). At 9, 15 and 60 dpi, whole lungs stained by X-gal. d, Immunofluorescence in 60 dpi lung sections with indicated antibodies. TI, TII indicate type I and II pneumocytes, respectively. Scale bar, 100 μm. PowerPoint slide

Figure 2. Conditional ablation of activated DASCp63/Krt5.

a, Modified Krt6a locus driving the human diphtheria toxin receptor and experimental scheme. DTox, diphtheria toxin. b, Immunofluorescence images of distal lung at 15 dpi with indicated diphtheria toxin (+/−DTox) condition. c, Quantification of Krt5+ and Krt6+ cells in DTox-treated mice. n = 2 mice per group, 6 sections covering the whole lung for each mouse. d, Lung sections from indicated mice following influenza virus infection and treatment with DTox. e, Morphometric analysis of interstitial densities in sections of lung (n = 3 mice per condition and time). Error bars, s.e.m. f, Anti-Pdpn (red) immunofluorescence of lung densities to reveal type I pneumocytes counterstained with DAPI (blue). Scale bar, 100 μm. H&E, haematoxylin and eosin. g, Peripheral capillary oxygen saturation (SpO2) values obtained by pulse oximetry (WT, n = 3, DTR ± DTox, n = 4) at indicated times. Error bars, s.e.m. **P < 0.01 for –DTox versus +DTox. h, Gene ontology classes and fold-change DASCp63/Krt5-ablation versus normal mouse lung at 30 dpi. Below, heat map of differentially expressed (P < 0.05) genes involved in pre-fibrosis, alveolar structure and vasculature. PowerPoint slide

Figure 3. Cloning and in vitro differentiation of DASCs and TBSCs.

a, Krt5+ cells in proximal (left) and distal (arrows, right) lung, with corresponding TBSC and DASC colonies (outside panels). Scale bar, 50 μm. b, Differentiation of TBSC and DASC in air–liquid interface cultures showing respectively stratified epithelia with ciliated (acetylated-Tub+) cells and a monolayer of differentiated cells. Scale bar, 20 μm. c, Unilaminar spheres formed by DASCs in three-dimensional Matrigel cultures and the expression of indicated markers in sections. Scale bar, 50 μm. Insets, high magnification. d, Scatter plot of gene expression of immature TBSCs and DASCs highlighting common and disparately expressed (fold change > 3, P < 0.001) genes. e, Scatter plot of gene expression of TBSC-ALI and DASC-MAT. f, Top, heat map of gene sets differentially expressed in murine alveoli and tracheal epithelium (P < 0.05). Bottom, heat map of differentially expressed genes in DASC-MAT versus TBSC-ALI (P < 0.05) informed by alveolar and tracheal data sets. g, Histogram of differentially expressed genes of DASC-MAT versus TBSC-ALI for which validating immunohistochemistry data are available (see http://www.proteinatlas.org/). PowerPoint slide

Figure 4. Transplantation of TBSClacZ and DASClacZ.

a, Schematic of pedigree generation and transplantation. 4OH-Tmx, 4-hydroxy-tamoxifen; ITD, intratracheal delivery. b, β-galactosidase activity in whole lung following DASClacZ transplantation. c, Comparison between β-galactosidase-positive (left panels) and -negative (right panels) regions of transplanted lung and markers of type I (1H8) and type II (SPC) cells. Scale bar, 50 μm. d, Heat map of selected, differentially expressed genes (P < 0.05) comparing immature DASClacZ before transplantation with laser-capture microdissected of lacZ-positive cells from transplanted lungs at 90 dpi. e, β-galactosidase activity in whole lung following TBSClacZ transplantation. f, From left, DASCGFP colony in culture; middle, cryosection of lung following DASCGFP transplantation. Scale bar, 50 μm. Right, immunofluorescence of anti-GFP, anti-Pdpn and anti-SPC in 40 dpi transplanted lung. Scale bar, 20 μm. PowerPoint slide

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.