Bronchoscopic isolation of clonogenic airway basal cells

In current study, we worked on the P63+/KRT5+ BCs in the airway epithelium of human lung which could possibly be the counterpart of mouse DASC. The workflow of BC isolation and expansion is summarized in Fig. 1A. Approximately 20,000–30,000 cells were brushed off from the luminal surface of donor’s 3rd–4th order bronchus using a 2-mm bronchoscopic brush (Wimberley et al., 1982) (Fig. 1B). The brushed-off cells were seeded onto embryo-derived feeder cells with the culture medium favoring BC growth (Zuo et al., 2015; Wang et al., 2015). After seeding 5,000 live cells onto 6-well plate, 9 (±2) cells grew up into visible tight colonies 3–5 days later with expression of human nucleus specific antigens, lung progenitor marker NKX2.1 and proliferation marker KI67 (Figs. 1C and S1A). All of the P0 colonies were confirmed epithelium origin (E-cadherin+, Fig. S1A) and stained double positive for airway basal cell markers KRT5 and P63 (Fig. 1C and 1D). We did not observe any P63 single positive colonies (Vaughan et al., 2015). Considering that BCs take for about 20% of total cell number in brushed samples of 3rd–4th order bronchus, it appeared that approximately 1% of the BCs in human airway could be clonogenic lung epithelium progenitors.

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 Full size image

We seeded one single BC onto feeder cells and grew them into one single colony which was then picked up by cloning cylinders and passaged continually. The latest passage of BC clones had gone through 50 doublings (=1015 fold expansion) in our lab. The single cell-derived BC clones and their original brush-off tissue samples were analyzed by high-throughput RNA sequencing (RNA-Seq). On average, we detected 16,230 genes and 25,223 transcripts. Thus, more than 60% of known human genes and transcripts were expressed in clonogenic BCs. Gene expression value correlation analysis showed that the clone transcriptome profiles are distinct from their original brush-off tissues, but the two clones from two independent persons share very similar transcriptome (Fig. 1E, Pearson correlation coefficient = 0.95). Single nucleotide polymorphism (SNP) analysis showed that BC clones have around 70% less polymorphism comparing to the brush-off tissues, which is in consistency with their single cell origination. High expression of BC markers (KRT5, P63, NGFR and S100A) and another putative mouse stem cell marker integrin α6β4 (Chapman et al., 2011) were observed in clones. In contrast, clonogenic BCs do not express other bronchial or alveolar lineage markers as shown by RNA-Seq and confirmed by immunostaining (Figs. 1F and S1B). Protein-protein interaction analysis of overexpressed genes indicated three major signal molecule networks including Notch1/2/3, FGF10/7 and Wnt7 ligand and their downstream components. All three signaling networks are previously known to play essential roles in embryonic lung development (Bellusci et al., 1997; Rajagopal et al., 2008; Tsao et al., 2008) (Fig. 1G). Gene ontology (GO) term analysis demonstrated critical biological processes enriched in BCs (Fig. 1H).

Clonal analysis of SOX9+ BCs

Importantly, RNA-Seq data also showed that clonogenic BCs highly express SOX9 (Sex Determing Region Y- Box 9), a transcriptional factor known to be enriched in branching tips of developmental lung. In embryonic development, SOX9 activity is required to maintain the undifferentiated status of distal lung progenitor and disruption of SOX9 function prevents adult alveoli formation (Perl et al., 2005; Rockich et al., 2013). Here we confirmed SOX9 expression in P63+/KRT5+ BC clones by immunostaining (Fig. 2A). Accordingly, by histological examination of human 2nd order (Fig. S2) and 3rd–4th order airway (Fig. 2B), we observed 1.3% ± 0.3% and 1.7% ± 0.5% SOX9-expressing P63+ BCs, respectively. The proportion of SOX9+ cells in total BCs is very close to our estimation in clonogenic assay as mentioned above (~1%), suggesting SOX9 as a marker to distinguish clonogenic BCs vs. other non-clonogenic BCs. Interestingly, we noticed that there are a few invaginations (rugaes) in 2–4 order human airway epithelium and the SOX9+ BCs are exclusively located near the base of the rugaes. There are averagely 3 (±1) SOX9+ BCs in each individual rugae. Further immunofluorescent examination showed a very small portion of them (<1%) are proliferative (KI67+) (Fig. 2C). Of note, SOX9+ BCs can also be isolated and expanded from those small airway (~1 mm diameter) samples, which is accessible by open-chest surgery or autopsy but not by bronchoscopy (data not shown).

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 Full size image

The whole brushing sampling and SOX9+ BCs cloning procedure was carried out on 15 individuals with a recovery rate of 100%. Donors are from 4 different disease categories including 5 normal healthy volunteers, 2 bronchiectasis patients, 3 chronic COPD patients, and 5 interstitial lung disease (ILD) patients with pulmonary fibrosis. The SOX9+ BCs from different categories of diseases showed no apparent difference in colony morphology (Fig. S3A) or marker expression (Fig. S3B). Their clonogenic efficiency seemed similar—but still need future investigation in much larger cohort to get statistically meaningful conclusion.

We further analyzed SOX9+ BCs at single cell resolution. 5 single cells from one person in normal group were selected at Passage 0 and expanded to Passage 1 and Passage 2. Great variation of their clonogenic capacity was observed at Passage 1 (coefficient variation = 59.9%) and Passage 2 (coefficient variation = 75.7%). Similar clonogenicity variation was observed in individuals from other disease categories and the average coefficient variation of all clones is 52.2% (Fig. S3C).

SOX9+ BCs grown on feeder cells can be transferred onto petri dish pre-coated with collagen fibers for feeder-free culture. The feeder-free cultured SOX9+ BC can also form colonies though their cell-cell contact within one colony is less tight comparing to those on feeders (Fig. 2D). The feeder-free cultured BCs are able to be passaged for at least 30 doublings with no obvious morphology change. Karyotyping indicated their stable genetic characteristics along with passaging (Fig. 2E). Quantitative analysis of progenitor markers (KRT5, P63 and SOX9) and lung epithelium lineage markers at both RNA and protein level indicated that there is no spontaneous differentiation of BCs in the culture process (Fig. 2F–H).

Xeno-transplanted SOX9+ BCs give rise to human lung in vivo

Next we examined whether the SOX9+ BC could differentiate and regenerate lung tissue by transplanting such cells into mouse lung parenchyma. Firstly, immunodeficient NOD-SCID mice were subjected to bleomycin intratracheal instillation, which lead to rapid onset (8 days after bleomycin) damage of centrilobular and surrounding regions as shown by microCT-scan and immunostaining. Masson trichrome staining for collagen and α-SMA immunostaining indicated severe tissue fibrosis of mouse lung at later time points (Zhang et al., 1996) (Fig. S5A–C). Scarce endogenous mouse p63+/Krt5+ distal airway stem cell expansion was observed in damaged lung parenchyma as reported previously (Vaughan et al., 2015) (Fig. S5D). Then we intratracheally delivered (Zuo et al., 2015) 1 × 106 GFP-labeled SOX9+ BCs into the injured mouse lung and analyzed the lung 3 weeks after transplantation. As shown in Fig. 3A, we observed large-scale incorporation of GFP+ human SOX9+ progenitors and their progeny into mouse lung. Direct fluorescence after tissue sectioning showed distribution of GFP+ human cells in mouse distal lung, some of them are morphologically indistinguishable from neighboring GFP− mouse lung structures (Fig. 3A). The chimerism of human-mouse lung was further confirmed by human-specific nucleus antigen Lamin A+C co-staining with GFP (Fig. 3B) and qPCR with human specific GAPDH primers (Fig. S6A). A few fully differentiated human cells have lost SOX9 marker expression and form air-sacs of similar size to mouse alveoli with AEC1 marker (AQP5 and HOPX) expression (Fig. 3C–E). Some transplanted GFP+ human cells could also incorporate into bronchiolar region of lung, where some of them gave rise to Club cell with CC10 marker expression while a few others became ciliated cells (acetylated-tubulin+, FOXJ1+), respectively (Fig. S6B–D). However, we hardly observed human SPC+ AEC2 in transplanted mouse lung. The differentiation potential of SOX9+ BCs was further confirmed by qPCR analysis of multiple marker genes with human specific primers. Both AEC1 and bronchiolar cell marker genes were strongly expressed in the chimera. For AEC2 marker genes, though SPB and LAMP3 were highly expressed, we did not detect SPC expression in the chimera, which was consistent with the immunostaining result (Fig. 3F).

Figure 3 Transplantated SOX9+ BCs regenerate functional human lung in 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 Full size image

In control experiment, we found that the transplanted SOX9+ progenitors cannot incorporate into non-injured healthy mouse lung or porcine pancreatic elastase-injured mouse lung (data not shown). Also, human lung-derived fibroblast cells (data not shown) or human cervix-derived P63+/KRT5+/SOX9+ progenitor cells (Figs. 3G and S6E) can barely incorporate into injured mouse lung either. This data indicated the tissue specificity of different adult stem/progenitor cells.

Regenerated lung by SOX9+ BC transplantation contributed to mouse pulmonary function

Functional alveolar unit requires close epithelium-capillary interaction for exchange of gas, energy and other substances. In the optically cleared mouse lung, we observed branching major blood vessels in transplanted mouse lung (Fig. S7A). We also found that the thin, long-shape human AEC1 aligned together with microvascular vessels which are positive for capillary endothelial markers CD34 and PECAM/CD31, with approximately 1 μm-thick integrinβ-1+ basement membrane between epithelium and capillary endothelium (Fig. 4A–C). And engrafted GFP+ human cells form adherens junctions and tight junctions with neighboring alveolar epithelial cells as shown by E-cadherin and ZO-1 staining on the border (Fig. 4D and 4E), which makes a closed space to maintain air pressure. In order to examine whether such blood-gas exchanging units are functionally connected with circulation, we developed a gold nanoparticle (AuNP) (Cheng et al., 2008)-based approach to mimic gas exchange and transport in vivo. The nanoparticles can be transported in blood and diffuse across cells (like O 2 and CO 2 ) due to its small size (~5 nm), water solubility and lipophilicity, and meanwhile can be detected by histology. One hour after injection of AuNPs into mouse tail vein, we detected significant gold signal in healthy mouse alveoli (Fig. S7B) as well as in GFP+ human alveoli (Fig. 4F), indicating the regenerated human tissues are functionally linked with circulation system. On the other side, after intratracheally aspiration of AuNPs, some GFP+ part of mouse lung showed significant gold signal, indicating the regenerated human tissues are anatomically linked with atmospheric air (Figs. 4G and S7C). As control, no or very little AuNPs signal was observed in damaged alveolar area by either way of particle delivery (Fig. S7B and S7C). These evidences implicated that the regenerated lung tissue has vascularized gas-exchange capacity, probably through recruitment of self-organizing capillary endothelial cells by SOX9+ BCs.

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 Full size image

We also found that SOX9+ BC transplantation effectively blocked the progression of mouse pulmonary fibrosis manifested as fibronectin accumulation and α-SMA positive myofibroblast expansion (Phan, 2012) in the human cell-enriched area (Fig. 5A and 5B), suggesting that regenerated human lung can replace damaged tissue in mouse model. Accordingly, alveoli regeneration by SOX9+ BC transplantation also improved the recipient mouse pulmonary function as shown by the decrease of CO 2 partial pressure, increase of O 2 partial pressure and O 2 saturation in artery blood (Fig. 5C–E).

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) CO 2 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) O 2 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) O 2 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 Full size image

TGF-β signaling modulates SOX9+ BC proliferation

To further improve the transplantation efficiency of SOX9+ BCs, we screened multiple drugs and found Pirfenidone, an FDA approved anti-pulmonary fibrosis drug (King et al., 2014) could facilitate the SOX9+ BC transplantation efficiency significantly. Interestingly, transforming growth factor-β (TGF-β) had the opposite effect (Figs. 6A and S8A). This discovery prompted us to study the underlying molecular and cellular mechanism. We found that Pirfenidone treatment can abolish TGF-β-induced phosphorylation of SMAD2/SMAD3 (Fig. 6B). In turn, TGF-β treatment significantly suppressed the clonogenicity and cell viability of SOX9+ BCs, which can be rescued by the SMAD2/SMAD3 inhibitor SB-431542 (Fig. 6C–E). Simutaneously, the expression of p15(INK4B), a G 1 cell cycle inhibitor, was strongly induced by TGF-β treatment together with mild change of some other cell cycle-related genes (Fig. 6F). TGF-β had little effect on the apoptosis of SOX9+ BCs (Fig. S8B). Collectively these experiments showed that the TGF-β/SMAD/P15 signaling axis could effectively modulate SOX9+ BC proliferation. Similar proliferation inhibitory effect of TGF-β/SMAD was recently reported on TBC as well (Mou et al., 2016).

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 Full size image

Autologous SOX9+ BCs transplantation clinical trial in bronchiectasis patients

Bronchiectasis is a chronic lung disease radiographically characterized by permanent pathologic dilation of the small and medium-sized bronchi, which may lead to respiratory failure and eventually to death. Patients with bronchiectasis, if left untreated, will have a continual decrease of their pulmonary function. Current pharmacological strategies to treat bronchiectasis such as antibiotics, mucolytics and anti-inflammatory agents could only control the disease exacerbation but not improve the pulmonary function nor repair the damaged lung tissue (ten Hacken et al., 2007). To explore the clinical feasibility of autologous SOX9+ BC transplantation, we conducted a pilot trial aiming to treat bronchiectasis by regenerating functional human lung. The general trial protocol and the cell manufacturer (Regend Therapeutics Co.Ltd) were archived by China Food and Drug Administration (CFDA) and National Health and Family Planning Commission of China, and the trial was performed in national approved stem cell clinical research institute (Southwest Hospital) after strict ethic commission review of preclinical data (A part of but not all preclinical data was released in the current manuscript).

Two patients diagnosed as non-CF bronchiectasis were firstly enrolled for autologous SOX9+ BC transplantation on April, 2016. Both patients are men in 50s, non-smokers. Patient 1 was diagnosed as bronchiectasis 8 years ago with productive cough and dyspnea on exertion symptom, which worsens continually under regular pharmacological treatment. CT scan shows multiple bronchial cylinder dilation and patchy consolidation in his lung. Patient 2 was diagnosed as bronchiectasis and COPD decades ago, with productive cough and dyspnea on exertion symptom, which worsens continually under regular pharmacological treatment. CT scan shows multiple bronchial cystic dilation, thicken bronchial wall and patchy consolidation in his lung.

For both patients, tissues were bronchoscopically collected from random region of left upper lobe and right upper lobe and transported to GMP (Good Manufacture Practices) level tissue culture facility for SOX9+ BC isolation and expansion (Fig. 7A). Isolated SOX9+ BCs were cultured on clinical-level feeder cells and then shifted to feeder-free culture condition. Totally 1 × 106/kg body weight of SOX9+ BCs were infused into distinct lobes of patients through bronchoscopy (Tzouvelekis et al., 2013) (Fig. 7B). Clinical status of patients was evaluated 1 day before and 1, 3 and 12 months after cell transplantation. Although it is almost impossible to directly track unlabelled transplanted cells in human, we did observe regional repair of cystic dilation after cell transplantation by high-resolution computed tomography (HRCT) scan for Patient 2 (Fig. 7C). The thickened bronchial wall also became thinner after cell therapy for Patient 2. Spirometry results indicated remarkable recovery of pulmonary function in both patients after transplantation as measured by FEV1, FVC and DLCO/VA (Fig. 7D). Importantly, no aberrant cell growth or other related adverse events were observed during the whole follow-up time. In the last follow-up (20 months after transplantation), Patient 1 described improvement of dyspnea, improvement of exercise capacity, less productive cough and less times of exacerbation after cell therapy; Patient 2 described less productive cough and less times of exacerbation after cell therapy. As it is well documented that bronchiectasis is a permanent, irreversible disease that cannot resolve spontaneously or with regular medicine, the recovery of patients suggested high probability that transplanted SOX9+ BCs were able to regenerate functional lung in human, which is consistent with our observation in animal models. And we will continue life-long observation on the two patients.