Regeneration of functional alveoli by adult human SOX9+ airway basal cell transplantation

Abstract

Irreversible destruction of bronchi and alveoli can lead to multiple incurable lung diseases. Identifying lung stem/progenitor cells with regenerative capacity and utilizing them to reconstruct functional tissue is one of the biggest hopes to reverse the damage and cure such diseases. Here we showed that a rare population of SOX9+ basal cells (BCs) located at airway epithelium rugae can regenerate adult human lung. Human SOX9+ BCs can be readily isolated by bronchoscopic brushing and indefinitely expanded in feeder-free condition. Expanded human SOX9+ BCs can give rise to alveolar and bronchiolar epithelium after being transplanted into injured mouse lung, with air-blood exchange system reconstructed and recipient's lung function improved. Manipulation of lung microenvironment with Pirfenidone to suppress TGF-β signaling could further boost the transplantation efficiency. Moreover, we conducted the first autologous SOX9+ BCs transplantation clinical trial in two bronchiectasis patients. Lung tissue repair and pulmonary function enhancement was observed in patients 3-12 months after cell transplantation. Altogether our current work indicated that functional adult human lung structure can be reconstituted by orthotopic transplantation of tissue-specific stem/progenitor cells, which could be translated into a mature regenerative therapeutic strategy in near future.

Keywords: alveoli; bronchiectasis; lung; regeneration; stem cell; transplantation.

Figures

Figure 1

Isolation and characterization of BCs from SOX9+human airway. (A) Diagram showing the process of clonogenic BCs isolation and expansion. (B) Bronchoscopic image showing brushing of cells from human airway. (C) Left, BC colonies grown on feeder cells; right, anti-KRT5 and anti-P63 immunostaining of BC colonies with nuclei counterstain. Human sample number n = 10. Scale bar, 100 μm. (D) Left, BCs in human airway by anti-KRT5 and anti-P63 immunostaining. Inset, high magnification with club cell (CC10+, cyan color) costaining; right, hematoxylin & eosin staining of the same section. Br, bronchus. Scale bar, 100 μm. (E) Heatmap showing transcriptome profile correlation value of BC clones and brush-off tissues. (F) Expression heatmap of selected, differentially expressed genes (P < 0.05) comparing BC clones and brush-off tissues. (G) Protein-protein interaction network of selected genes with high expression level in BC clones. (H) Enriched gene ontology classes of BC clones versus brush-off tissues

Figure 2

Feeder-free expansion of SOX9+ BCs. (A) Immunostaining of SOX9+ BCs with anti-P63, anti-KRT5 and anti-SOX9 antibodies. (B) SOX9+ BCs in rugae of 3rd order human airway by anti-SOX9, anti-P63 and anti-CC10 immunostaining. Scale bar, 100 μm. (C) SOX9+ BCs in rugae of 3rd order human airway by anti-KI67 immunostaining. (D) BC colony cultured on feeder-free condition. (E) Karyotyping of cultured BCs. (F) qPCR showing alveolar and bronchial epithelium marker gene expression of human lung sample and SOX9+ BCs in early (P2) and late (P8) passages. n = 3, biological replicates. Error bars, S.E.M. (G) qPCR showing progenitor cell marker (Krt5, P63 and SOX9) gene expression of human lung sample and SOX9+ BCs in early (P2) and late (P8) passages. n = 3, biological replicates. Error bars, S.E.M. (H) Western blotting showing marker gene expression of human lung sample and SOX9+ BCs in early (P2) and late (P8) passages

Figure 3

Transplantated SOX9+ BCs regenerate functional human lungin vivo. (A) Left, direct fluorescence image under stereomicroscope showing NOD-SCID mouse lung without (upper panel) or with (lower panel) GFP-labeled SOX9+ BC transplantation. Right, cryo-section and direct fluorescence imaging of transplanted GFP-labeled SOX9+ BCs in lung parenchyma. Scale bar, 100 μm. (B) Immunofluorescence imaging of transplanted GFP-labeled SOX9+ BCs in lung parenchyma with human specific Lamin A+C marker costaining. (C) Fully differentiated GFP+ cells lost SOX9 marker expression (arrowhead indicated). Scale bar, 10 μm. (D) Confocal image with human specific Lamin A+C immunostaining (HuLamin) showing regenerated type I (AQP5+) alveolar cells. No type II (SPC+) cells were observed. (E) Confocal image showing regenerated AEC1 (AQP5+ and HOPX+). AQP5 as a membrane-bound protein distributes on surface of GFP+ cells. Arrowheads indicated the overlay of HOPX with GFP signal in nucleus. Scale bar, 20 μm. (F) qPCR with human specific primers showing alveolar and bronchiolar epithelium marker gene expression in SOX9+ BC transplanted chimeric lung (AEC1: AQP5 and HOPX; AEC2: SPB and LAMP3; bronchiolar cells: SCGB1A1 and MUC1). Biological replicates, n = 3. Error bars, S.E.M. (G) Left, clonogenic BCs isolated from human cervix epithelium obtained by biopsy. Right, transplantation of equal numbers of BCs from lung and cervix indicated different incorporation efficiency

Figure 4

Regenerated alveoli with functional epithelium-capillary system. (A) Transplanted SOX9+ BCs (anti-GFP) and capillary endothelium marker (anti-CD34). Scale bar, 100 μm. (B) Confocal image of SOX9+ BCs regenerated alveoli (Alv) and the neighboring capillary blood vessel (Bv). Left, immunofluorescence; right, bright field. Scale bar, 20 μm. (C) Confocal image showing the basement membrane (ITGB1+, white color, arrowhead indicated) between regenerated alveoli epithelium and capillary endothelium (CD31+). Scale bar, 10 μm. (D) Confocal image showing the cell adherens junction (E-cadherin+, white color) between regenerated alveoli epithelial cells. Scale bar, 20 μm. (E) Confocal image showing the cell tight junction (ZO-1+) between regenerated alveoli epithelial cells. Scale bar, 20 μm. (F) Direct fluorescence image of the transplanted GFP-labeled SOX9+ BCs (green) and bright-field image of tail vein delivered gold nanoparticles (AuNPs) of the same region (brown). Scale bar, 100 μm. (G) Direct fluorescence image of the transplanted GFP-labeled SOX9+ BCs (green) and bright-field image of intratracheally delivered gold nanoparticles (AuNPs) of the same region (brown). Scale bar, 100 μm

Figure 5

BC transplantation rescued mouse pulmonary function. (A) Injured mouse lung without or with GFP-labeled SOX9+ BCs transplantation by anti-GFP and anti-Fibronectin co-staining. Scale bar, 200 μm. (B) Left, immunofluorescence image of injured mouse lung transplanted with GFP-labeled SOX9+ BCs; right, immunostaining on the same section showing exclusion of α-SMA+ myofibroblasts from GFP+ area. Scale bar, 200 μm. (C) CO2 partial pressure of mouse arterial blood before and 1 month after bleomycin-induced injury with or without SOX9+ BCs transplantation. Each dot indicates an individual mouse. (D) O2 partial pressure of mouse arterial blood 1 month after bleomycin-induced injury with or without SOX9+ BCs transplantation. Each dot indicates an individual mouse. (E) O2 saturation of mouse arterial blood before and 1 month after bleomycin-induced injury with or without SOX9+ BCs transplantation. Each dot indicates an individual mouse

Figure 6

TGF-β signaling modulates SOX9+BC proliferation. (A) Direct fluorescence image of mouse lung transplanted with 1 × 106 GFP-labeled SOX9+ BCs under dissection microscope. Each lung was from mouse with indicated treatment and harvested 7 days after transplantation. The left lobes were analyzed and the GFP+ cell numbers (×106) were counted by flow cytometry analysis. Biological replicates, n = 3. PFD, Pirfenidone. (B) SOX9+ BCs were stimulated with 10 ng/mL TGF-β for 2 h, with or without 1 mg/mL Pirfenidone treatment overnight. Western blotting of cell lysates with anti-phosphated-Smad2/3 and anti-total Smad2/3 antibodies was performed to examine the activation of TGF-β pathway. (C) Direct fluorescence imaging of GFP-labeled SOX9+ BCs cultured in a 6-well plate in the absence or presence of 10 ng/mL TGF-β. Scale bar, 200 μm. (D) Quantification of clonogenicity of SOX9+ BCs in the presence of 10 ng/mL TGF-β or 10 mmol SB. SB, TGF-β type I receptor inhibitor SB-431542. Technical replicates n = 3. (E) WST viability assay of SOX9+ BCs treated by 10 ng/mL TGF-β or 5 mmol TGF-β inhibitor SB-431542, or their combination. Technical replicates n = 3. (F) qPCR showing cell cycle-related gene expression level of SOX9+ BCs with 10 ng/mL TGF-β treatment for indicated h. Biological replicates, n = 3

Figure 7

Autologous SOX9+ BC transplantation: a pilot clinical study. (A) Cultured SOX9+ BCs from two patients of bronchiectasis. (B) Diagram showing the number of SOX9+ BCs (×107) transplanted into each lobe of Patient 1/Patient 2. Five lobes are labeled with different colors. Note the Patient 1 undertook left lower lobectomy a decade ago. (C) Representative images of consecutive CT scan show the regional recovery of bronchiectasis (yellow square) 1 year after autologous SOX9+ BC transplantation in Patient 2. (D) Measurement of pulmonary function and exercise capacity in both patients: FEV1 (forced expiratory volume in 1 second), FVC (forced vital capacity), DLCO/VA (diffusing capacity of the lung for carbon monoxide adjusted by the alveoli volume) were shown as absolute value and percentage to predicted level. For each parameter, percentage >80% was regarded as clinically normal