Cloned airway basal progenitor cells to repair fibrotic lung through re-epithelialization

Abstract

Irreversible damage of the lung epithelium in idiopathic pulmonary fibrosis (IPF) patients causes high mortality worldwide, with no lung repair approaches available currently. Here we show that in murine and monkey models, the KRT5⁺ P63⁺ progenitor cells in airway basal layer can enter the alveolar area post fibrotic injury. Aided with an automated culture system, we clone and characterize airway basal progenitor cells from 44 donors with various lung conditions. Transplantation of human progenitor cells into the mouse lung efficiently re-epithelializes the injured alveolar area, forms new respiratory tract and saccule-like structures, which ameliorates fibrotic lesions and improves survival of mice. Mechanistically, the engrafted human progenitor cells do not function by differentiating into mature alveolar cells in mouse lung; instead, they differentiate into saccular cells expressing multiple tight junction proteins such as CLDN4, which help the lung to re-establish epithelial barriers. Furthermore, by cloning P63⁺ airway basal progenitors from larger mammals and birds, we construct multiple lung-chimerism animals and uncover the evolutionarily conserved roles of these progenitor cells in lung repair. Overall, our data highlight the fate of airway basal progenitor cells in fibrotic lung and provide a potential therapeutic strategy for pulmonary diseases that lack inherent recovery mechanisms.

Fig. 1. Analysis of airway basal progenitors in monkey and human.

a Representative images of consecutive CT scans showing the presence of local lesions (red arrow) before a 71 days post first bleomycin injured in the macaque. b Masson’s trichrome staining for collagen deposition (blue) on histological section of macaque monkey lung 71 days post bleomycin injury. Scale bar, 80 μm. c Immunofluorescence images of KRT5⁺ pods in monkey lung injured by bleomycin with anti-KRT5 (red) staining and DNA counterstain (DAPI, blue). Scale bar, 100 mm. d Quantification of macaque KRT5⁺ cells numbers in the alveolar area of control or injured macaque monkey lung (n = 6 technical replicates). Data are presented as mean ± SEM. Statistical analysis was performed using a two-tailed t test (***P < 0.001). e Colony forming numbers of macaque KRT5⁺ P63⁺ cells derived from control or injured macaque monkey lung by in vitro culture (n = 3 biological replicates). Data are presented as mean ± SEM. Statistical analysis was performed using a two-tailed t test (****P < 0.0001). f Representative lung CT images of sampling and damaged area of the IPF patients. Green circle: sampling area; red circle: damaged area. g Left, Brightfield image of human airway basal progenitor cells clones. Right, immunostaining of airway basal progenitor cell clones with anti-KRT5 (green), anti-P63 (red) and DNA counterstain (DAPI, blue). Scale bar, 100 μm. h The automated progenitor cell culture system. Left, an overview of the system; Right, a 6-axis robotic arm mimicking the human arm movements. i Representative images of human airway basal progenitor cells cultured by junior workers or by automated cell culture system. Scale bar,100 μm. j Unsupervised principal component analysis (PCA) of whole genome transcriptome of airway basal progenitor cells derived from healthy donors or patients. Each dot represented the cell clone derived from an individual donor. The dotted line indicated the clones which were transcriptomically similar to healthy donors. k Correlation analysis of gene expression level with pulmonary function of patients. The shaded area represents the 95% confidence interval around the regression line, reflecting the standard error of the fitted values.

Fig. 2. Transplanted human airway basal progenitors re-epithelialized the injured mouse lung.

a Direct fluorescence image of mouse lung transplanted with human GFP-labeled human airway basal progenitor cells showing engraftment, captured under a stereomicroscope. Scale bar, 1 mm. b Immunofluorescence images of engrafted human cells in mouse lung with human-specific antibodies anti-HuLamin (red) immunostaining. Nuclei counterstain, DAPI (blue). Scale bar, 1 mm. c IHC staining of engrafted cells (brown) in mouse lung 8 (left) or 35 days (right) post-transplantation (dpt) with anti-GFP antibodies. Scale bar, 1 mm. Enlarged inset: high magnification views of the representative pod and saccular structures. Scale bar, 100 μm. d Mean linear intercept (MLI) of native human (hAlveoli) and murine alveoli (mAlveoli) as well as human saccules (Sac) (n = 13, 6, 23 technical replicates from 3 biological replicates). Data are presented as mean ± SEM. e Wall thickness of native human (hAlveoli) and murine alveoli (mAlveoli) as well as human saccules (Sac) (n = 50, 50, 275 technical replicates from 3 biological replicates). Data are presented as mean ± SEM. f 3D visualization showing the connection of human pods and saccules(sac) with mouse airway. 3D reconstruction was performed based on anti-GFP IHC stained serial sections of mouse lungs, which were transplanted with human airway basal progenitor cells and analyzed on 8 dpt (left) or 35 dpt (right), respectively. g Schematic diagram showing the process of lung respiratory tract growth and saccule formation from mouse airway. h H&E staining (left) and anti-GFP IHC staining (right) of engrafted human cells on a lung tissue section for spatial transcriptomic analysis. Green circle: saccule zone, blue circle: pod zone. Scale bar, 300 μm. i Spatial visualization of human cells in lung section which was noted using human reference genome (GRCh38) by spatial transcriptomic analysis. Scale bar, 1 mm. j Spatial visualization of various gene expression in lung sections by spatial transcriptomic analysis. Green circle: saccule zone, blue circle: pod zone. Scale bar, 300 μm.

Fig. 3. Functional analysis of human airway basal progenitor cells in vivo.

a Spatial visualization of the mouse (red) and human (green) epithelial cell marker CDH1 (E-cadherin) and CLDN4 gene expression in different lung zones. Yellow circle: damaged honeycombing zone; red circle: normal alveolar tissue zone; green circle: saccular zone; blue circle: pod zone. Scale bar, 1 mm. b Spatial visualization of mouse fibrotic marker Collagen1 (Col1a1) gene expression in different lung zones. High magnification indicated the Collagen1 signal in pod and saccule zones. Scale bar, 1 mm. c Expression level of multiple mouse marker genes in different lung zones. Normal, normal alveolar area; Damage, damaged honeycombing zone; Sac, saccular zone (n = 5 technical replicates). d Immunofluorescence images of mouse capillary vessels in the saccular zone with anti-GFP (green), anti-CD31 (red), and anti-CD34 (red) antibodies co-staining and DNA counterstain (DAPI, blue). Scale bar, 50 μm. e Barrier function assays of airway basal progenitor cells in vitro. Left, schematic diagram of the barrier function transwell assays; middle, TEER assay of monolayers formed by airway basal progenitor cells or A549 cells before and after Triton X-100 treatment (n = 6 biological replicates); right, assay of fibroblast migration through monolayers formed by airway basal progenitor cells or A549 cells (n = 6 biological replicates). f Quantification of the inflammatory area of mouse lung with or without airway basal progenitor cell transplantation (n = 8, 12, 15 technical replicates from 3 biological replicates). g Ashcroft score of lung fibrosis and quantification of the relative fibrotic area by Masson’s trichrome staining of mouse lungs with or without airway basal progenitor cell transplantation (n = 8, 12, 15 technical replicates from 3 biological replicates). h O₂ partial pressure to CO₂ partial pressure ratio of mouse arterial blood with or without human airway basal progenitor cell transplantation (n = 9 biological replicates). i Survival curve of mice challenged by bleomycin with or without airway basal progenitor cell treatment (n = 3, 4, 5 biological replicates). Statistical analysis was performed using the Log-rank (Mantel-Cox) test. c, e–h All data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test and two-way ANOVA with Sidak’s multiple comparisons test (for the middle panel of E) (**P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, no significant difference).

Fig. 4. Fate mapping of human airway basal progenitor cells in vivo.

a Single-cell RNA-sequencing cellular cluster map of GFP⁺ human cell from 8 (left) and 35 (right) dpt mouse lungs. The pie plots showed the percentages of distinct cell clusters. b Feature plots showing the expression of multiple marker genes in distinct single-cell clusters of engrafted human cells. c Feature plots showing the expression of genes signatures in distinct single-cell clusters of engrafted human cells. d Immunofluorescence images of engrafted human cells with anti-GFP (green), ciliated cell marker anti-acetylated-tubulin (red), Club cell marker anti-CC10 (red) or tight junction marker anti-CLDN4 (red) antibodies staining. Scale bar, 50 μm. e Gene Ontology enrichment analysis of the differentially expressed genes identified in CLDN4⁺ CLDN3⁺ saccular cells. f Pseudo-time trajectory projected onto a UMAP of transplanted human cells using the quiescent KRT5⁺ P63⁺ airway basal progenitors as a starting point. Pseudo-time values are color-coded. g Density of each human cell population ordered by pseudo-time as computed by reversed graph embedding approach of Monocle3. h Schematic diagram showing the putative differentiation paths starting from KRT5⁺ P63⁺ airway basal progenitor cells predicted by the pseudo-time analysis. i Schematic diagram showing the putative re-epithelialization process in the fibrotic lung after human airway basal progenitor transplantation.

Fig. 5. Clone airway P63⁺ cells from more mammals and birds for xeno-transplantation.

a Brightfield and direct fluorescence images of mouse lungs post-transplantation with GFP-labeled mouse, goat, porcine, and canine airway basal progenitor cells. Scale bar, 1 mm. b Cell engraftment efficiency of goat, porcine, and canine clones in mouse lung 8 days and 35 days post-transplantation (n = 3). Data are presented as mean ± SEM. Statistical analysis was performed using two-tailed t tests to compare the 8dpt and 35dpt groups within each species. (*P < 0.05, **P < 0.01, ***P < 0.001). c Engrafted goat cells in mouse lungs by anti-GFP (green) immunostaining with DNA counterstain (DAPI, blue). Upper, without cell transplantation; lower, with goat airway basal progenitor cell transplantation. Scale bar, 1 mm. d 3D reconstruction of anti-GFP IHC stained serial sections of mouse lung, which were transplanted with goat airway basal progenitor cells (red) and analyzed on 35 dpt. The blue color indicated mouse bronchi, red color indicated goat cells. e Immunofluorescence images of engrafted goat cells in mouse lung by anti-GFP (green), anti-AQP5 (red) immunostaining, and DNA counterstain (DAPI, blue). Scale bar, 100 μm. f H&E staining of the native goat alveoli section. Scale bar, 100 μm. g Quantification of the inflammatory area of mouse lung with or without goat airway basal progenitor cell transplantation (n = 4, 6, 6 technical replicates from 3 biological replicates). h–j Arterial blood gas analysis of murine arterial blood with or without goat airway basal progenitor cell transplantation. Each dot indicated an individual mouse (n = 3, 4, 4 biological replicates). g–j All data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, no significant difference). k H&E staining of the native pigeon alveoli section. Left, scale bar, 40 μm. Right, scale bar, 80 μm. l Immunofluorescence images of native pigeon lung with anti-α-SMA (green), and air capillary marker anti-CK7 (red) immunostaining and DNA counterstain (DAPI, blue). Scale bar, 50 μm. m Immunofluorescence images of native pigeon airway with anti-KRT8 (green), anti-P63 (red) staining, and DNA counterstain (DAPI, blue). Scale bar, 50 μm. n Immunofluorescence images of engrafted pigeon cells in mouse lungs with bird-specific antibody anti-chicken α-Actin (green) and air capillary marker anti-CK7 (red) co-staining. Scale bar, 20 μm.