Kate H.C. Gowers

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

RATIONALE Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. OBJECTIVES To define a scalable cell culture system to deliver airway epithelium to clinical grafts. METHODS Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. MEASUREMENTS AND MAIN RESULTS 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. CONCLUSIONS Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

Mesenchymal stromal cell delivery of full-length tumor necrosis factor-related apoptosis-inducing ligand is superior to soluble type for cancer therapy.

Background aims Mesenchymal stromal cell (MSC) delivery of pro-apoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an attractive strategy for anticancer therapy. MSCs expressing full-length human TRAIL (flT) or its soluble form (sT) have previously been shown to be effective for cancer killing. However, a comparison between the two forms has never been performed, leaving it unclear which approach is most effective. This study addresses the issue for the possible clinical application of TRAIL-expressing MSCs in the future. Methods MSCs were transduced with lentiviruses expressing flT or an isoleucine zipper-fused sT. TRAIL expression was examined and cancer cell apoptosis was measured after treatment with transduced MSCs or with MSC-derived soluble TRAIL. Results The transduction does not adversely affect cell phenotype. The sT-transduced MSCs (MSC-sT) secrete abundant levels of soluble TRAIL but do not present the protein on the cell surface. Interestingly, the flT-transduced MSCs (MSC-flT) not only express cell-surface TRAIL but also release flT into medium. These cells were examined for inducing apoptosis in 20 cancer cell lines. MSC-sT cells showed very limited effects. By contrast, MSC-flT cells demonstrated high cancer cell-killing efficiency. More importantly, MSC-flT cells can overcome some cancer cell resistance to recombinant TRAIL. In addition, both cell surface flT and secreted flT are functional for inducing apoptosis. The secreted flT was found to have higher cancer cell-killing capacity than either recombinant TRAIL or MSC-secreted sT. Conclusions These observations demonstrate that MSC delivery of flT is superior to MSC delivery of sT for cancer therapy.

TRAIL delivery by MSC-derived extracellular vesicles is an effective anticancer therapy

ABSTRACT Extracellular vesicles (EVs) are lipid membrane-enclosed nanoparticles released by cells. They mediate intercellular communication by transferring biological molecules and therefore have potential as innovative drug delivery vehicles. TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of cancer cells. Unfortunately, the clinical application of recombinant rTRAIL has been hampered by its low bioavailability and resistance of cancer cells. EV-mediated TRAIL delivery may circumvent these problems. Mesenchymal stromal cells (MSCs) produce EVs and could be a good source for therapeutic EV production. We investigated if TRAIL could be expressed in MSC-derived EVs and examined their cancer cell-killing efficacy. EVs were isolated by ultracentrifugation and were membranous particles of 50–70 nm in diameter. Both MSC- and TRAIL-expressing MSC (MSCT)-derived EVs express CD63, CD9 and CD81, but only MSCT-EVs express surface TRAIL. MSCT-EVs induced apoptosis in 11 cancer cell lines in a dose-dependent manner but showed no cytotoxicity in primary human bronchial epithelial cells. Caspase activity inhibition or TRAIL neutralisation blocked the cytotoxicity of TRAIL-positive EVs. MSCT-EVs induced pronounced apoptosis in TRAIL-resistant cancer cells and this effect could be further enhanced using a CDK9 inhibitor. These data indicate that TRAIL delivery by MSC-derived EVs is an effective anticancer therapy.

Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3–8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs.

Co-culture-expanded human basal epithelial stem cells for application in tracheal tissue engineering

Abstract Background Stem-cell-based tracheal replacement has been used to treat patients with end-stage airway disease in compassionate cases. Clinical experience suggests that restoration of an epithelial barrier is a priority to improve outcomes, but recent data suggest that a substantial number of autologous epithelial cells are required for effective engraftment. Existing cell culture methods fail to expand the airway epithelial progenitor pool, presenting a considerable hurdle for progression to clinical trials. We aimed to assess feeder layers and a Rho-associated kinase inhibitor as a method to expand human respiratory epithlieum for airway bioengineering. Methods Human epithelial cells from endobronchial biopsy samples were cultured on mouse 3T3-J2 fibroblast feeder layers in medium containing Y-27632, a Rho-associated kinase inhibitor (3T3+Y). Air-liquid interface and tracheosphere assays were done to determine differentiation capacity, and high-speed video microscopy to observe ciliary behaviour. Telomerase expression was measured with immunofluorescence and western blotting and telomere length with real-time PCR. Cells were karyotyped and engraftment potential tested in ex-vivo and in-vivo xenograft models. Findings 3T3+Y allowed for rapid and sustained expansion of airway epithelial basal cells. Population doublings demonstrated that cultures derived from donor biopsy samples could provide sufficient cell numbers to recellularise human tracheal scaffolds. At clinically applicable passages, cells were capable of airway differentiation in vitro, forming both ciliated and goblet lineages. Ciliary beat frequency and beat pattern were within normal range. Cells were karyotypically normal despite extensive expansion. Protein expression of telomerase was increased in 3T3+Y compared with that in conventional growth medium, and telomere length appeared to stabilise. Cultured cells repopulated a decellularised tracheal scaffold ex vivo and restored a differentiated epithelium in an in-vivo tracheal transplantation xenograft model. Interpretation The findings show that 3T3+Y generates large numbers of airway basal epithelial stem cells that retain qualities desirable for clinical transplantation. These data have important implications for personalised autologous airway epithelial cell therapy because cells could be obtained rapidly and in an appropriate number. Preclinical and clinical validation is required. Funding Wellcome Trust.

Optimized isolation and expansion of human airway epithelial basal cells from endobronchial biopsy samples

Autologous airway epithelial cells have been used in clinical tissue‐engineered airway transplantation procedures with a view to assisting mucosal regeneration and restoring mucociliary escalator function. However, limited time is available for epithelial cell expansion due to the urgent nature of these interventions and slow epithelial regeneration has been observed in patients. Human airway epithelial cells can be expanded from small biopsies or brushings taken during bronchoscopy procedures, but the optimal mode of tissue acquisition from patients has not been investigated. Here, we compared endobronchial brushing and endobronchial biopsy samples in terms of their cell number and their ability to initiate basal epithelial stem cell cultures. We found that direct co‐culture of samples with 3T3‐J2 feeder cells in culture medium containing a Rho‐associated protein kinase inhibitor, Y‐27632, led to the selective expansion of greater numbers of basal epithelial stem cells during the critical early stages of culture than traditional techniques. Additionally, we established the benefit of initiating cell cultures from cell suspensions, either using brushing samples or through enzymatic digestion of biopsies, over explant culture. Primary epithelial cell cultures were initiated from endobronchial biopsy samples that had been cryopreserved before the initiation of cell cultures, suggesting that cryopreservation could eliminate the requirement for close proximity between the clinical facility in which biopsy samples are taken and the specialist laboratory in which epithelial cells are cultured. Overall, our results suggest ways to expedite epithelial cell preparation in future airway cell therapy or bioengineered airway transplantation procedures.

Expansion of airway basal epithelial cells from primary human non-small cell lung cancer tumors: 3T3+Y for NSCLC cell culture

Pre‐clinical non‐small cell lung cancer (NSCLC) models are poorly representative of the considerable inter‐ and intra‐tumor heterogeneity of the disease in patients. Primary cell‐based in vitro models of NSCLC are therefore desirable for novel therapy development and personalized cancer medicine. Methods have been described to generate rapidly proliferating epithelial cell cultures from multiple human epithelia using 3T3‐J2 feeder cell culture in the presence of Y‐27632, a RHO‐associated protein kinase (ROCK) inhibitor, in what are known as “conditional reprograming conditions” (CRC) or 3T3 + Y. In some cancer studies, variations of this methodology have allowed primary tumor cell expansion across a number of cancer types but other studies have demonstrated the preferential expansion of normal epithelial cells from tumors in such conditions. Here, we report our experience regarding the derivation of primary NSCLC cell cultures from 12 lung adenocarcinoma patients enrolled in the Tracking Cancer Evolution through Therapy (TRACERx) clinical study and discuss these in the context of improving the success rate for in vitro cultivation of cells from NSCLC tumors.

Use of a decellularised dermis scaffold and human bronchial epithelial cells to tissue engineer airway mucosa suitable for tracheal transplantation

Abstract Background Tracheal reconstruction relies on the use of a split skin graft to re-epithelialise the mucosal layer. Since split skin grafts are made up of a keratinising stratified epithelial layer, sloughing occurs within the airway with mucus retention and subsequent airway obstruction. The delivery of a graft with the same mucociliary function as the native airway would overcome these limitations and greatly improve the safety and effectiveness of this type of surgery. We aimed to generate a transplantable tissue-engineered respiratory epithelial graft with mucociliary function. Methods Cadaveric human skin was decellularised and the epidermal layer removed. Human bronchial epithelial cells were seeded with human respiratory fibroblasts onto the dermis at densities of 1 × 10 6 per cm 2 and 1 × 10 4 per cm 2 , respectively, and cultured at air–liquid interface in a transwell system. At 3 weeks, the constructs were transplanted onto a decellularised trachea that had been prevascularised within a muscle wrap in an immunosuppressed New Zealand White rabbit. Findings After 3 weeks of air–liquid interface culture, high-speed video microscopy showed beating cilia on the surface of the dermis, and the epithelial layer stained positively for the ciliated cell marker acetylated α-tubulin, the secretory cell marker MUC5AC, and the epithelial cell marker pan-cytokeratin on top-down whole-mount confocal microscopy. Staining with haematoxylin and eosin (H&E) demonstrated a pseudostratified mucociliary layer along the length of the dermis. 24 h after transplantation, a pseudostratified, ciliated layer could be observed on H&E staining of sections of trachea. At 5 days, the respiratory epithelial layer consisted of a single layer of cytokeratin 5-positive epithelial cells. Interpretation This study is the first, to our knowledge, to report the delivery of a transplantable tissue-engineered respiratory epithelial graft with mucociliary function. 24 h after transplantation the mucociliary layer was preserved although only a basal layer was demonstrated by 5 days, possibly due to the loss of the air–liquid interface within the muscle wrap. Funding Medical Research Council.

Use of a collagen I scaffold with embedded respiratory fibroblasts and Rho kinase inhibitor to tissue-engineer airway mucosa

Abstract Background Techniques to replace airway mucosa rely on split skin or buccal mucosal grafts. These grafts lead to sloughing within the airway, mucus retention, and infection because of the absence of ciliary function. To address this problem, we have tissue-engineered a respiratory epithelial sheet to replace damaged airway mucosa. This type of graft has the potential to greatly improve the safety and effectiveness of airway surgery including the emerging technique of tissue-engineered tracheal transplantation. Methods Respiratory epithelial cells and fibroblasts were expanded from tracheal biopsy samples. We used a Cyquant DNA dye (Thermo Fisher, Waltham, MA, USA), BrdU ELISA (Roche, Burgess Hill, UK), and Live/Dead (Thermo Fisher, Waltham, MA, USA) assays to determine attachment, proliferation, and cell survival, respectively. Collagen I scaffolds were fabricated by dehydrating collagen I hydrogel with or without fibroblasts using RAFT insert absorbers (Lonza, Slough, UK). Epithelial cells were cultured at an air-liquid interface in a transwell system after 7 days of submerged culture with media containing a Rho kinase inhibitor (Y-27632). Findings Collagen I led to significantly greater levels of epithelial attachment compared with fibronectin, laminin 5, and vitronectin (ANOVA, Sidaks multiple comparison test, p=0·0006) with no difference in BrdU expression after 24 h of culture. The addition of Y-27632 increased BrdU expression in epithelial cells within submerged culture at 24 h and shortened time to confluency. Live/Dead assay demonstrated fibroblast survival within the scaffold up to 3 weeks and improved epithelial attachment in the fibroblast-containing scaffolds. Evidence of ciliation was detected at 3 weeks in the fibroblast-embedded scaffolds, and the scaffold could be easily detached and sutured to replace an area of lost mucosa in a dermal wound model. Interpretation We have found that collagen I is the most suitable matrix protein in terms of epithelial attachment and proliferation; and after dehydration it delivers a mechanically stable scaffold. The ability to embed fibroblasts delivers a more biomimetic arrangement that improves epithelial cell attachment and is important in differentiation in long-term culture. Y-27632 should be included to increase epithelial cell expansion during the submerged proliferative phase. Funding Medical Research Council.

Role of LRIG1-dependent EGFR signalling on pathway inhibition in airway homoeostasis and lung cancer development

Abstract Background LRIG1 is a negative regulator of epidermal growth factor receptor (EGFR) signalling, and regulates stem-cell compartments in skin and gut epithelial cells. Interestingly, LRIG1 is lost in samples from patients with preinvasive squamous cell lung cancer. Basal cells are the upper airway stem cells and the putative origin of this cancer. We aimed to delineate LRIG1 expression within the airway, its effect on the stem-cell compartment, and whether its loss causes preinvasive disease. Methods Lrig1 (EGFP-IRES-CreERT2) mice were used to determine LRIG1 expression in the airways with immunofluorescence and flow cytometry. Basal stem-cell expression of LRIG1 and correlation with cell proliferation were examined. LRIG1+ cells were isolated with flow cytometry, and colony-formation and spheroid-formation assays were used to compare LRIG1+ and LRIG1– basal cells. To lineage trace LRIG+ cells, Lrig1 mice were crossed with reporters and were transgene activated. In-vivo and ex-vivo studies were performed. Data are given as mean (SE). Findings LRIG1 expression was detected throughout the airway epithelium and within 66·3% (3·01) of basal cells. This LRIG1+ subpopulation of basal cells showed increased proliferation compared with LRIG1– cells (12·52% [1·82] vs 6·27 [2·13], p=0·0156). When plated in colony-formation assays, flow-sorted LRIG1+ expressing basal cells showed increased colony-forming efficacy and also had a greater spheroid-forming capacity (spheroid-forming capacity 3·3% [0·7] vs 0·56 [0·26], p=0·031). Ex-vivo lineage tracing assessed with spheroid assays indicated the development of clonal patches derived from LRIG+ basal cells. Formation of in-vivo clonal patches also occurred. Interpretation Our initial data indicate that LRIG1 has a role in airway stem-cell progenitor regulation and that it marks a more proliferative basal cell population. These data highlight the need to further investigate how the loss of LRIG1 leads to epithelial dysregulation and development of malignancy in patients. Funding The Wellcome Trust, Cancer Research UK.