Inflammatory Activity of Epithelial Stem Cell Variants from Cystic Fibrosis Lungs Is Not Resolved by CFTR Modulators.

Rationale: CFTR (cystic fibrosis transmembrane conductance regulator) modulator drugs restore function to mutant channels in patients with cystic fibrosis (CF) and lead to improvements in body mass index and lung function. Although it is anticipated that early childhood treatment with CFTR modulators will significantly delay or even prevent the onset of advanced lung disease, lung neutrophils and inflammatory cytokines remain high in patients with CF with established lung disease despite modulator therapy, underscoring the need to identify and ultimately target the sources of this inflammation in CF lungs. Objectives: To determine whether CF lungs, like chronic obstructive pulmonary disease (COPD) lungs, harbor potentially pathogenic stem cell "variants" distinct from the normal p63/Krt5 lung stem cells devoted to alveolar fates, to identify specific variants that might contribute to the inflammatory state of CF lungs, and to assess the impact of CFTR genetic complementation or CFTR modulators on the inflammatory variants identified herein. Methods: Stem cell cloning technology developed to resolve pathogenic stem cell heterogeneity in COPD and idiopathic pulmonary fibrosis lungs was applied to end-stage lungs of patients with CF (three homozygous CFTR:F508D, one CFTR F508D/L1254X; FEV1, 14-30%) undergoing therapeutic lung transplantation. Single-cell-derived clones corresponding to the six stem cell clusters resolved by single-cell RNA sequencing of these libraries were assessed by RNA sequencing and xenografting to monitor inflammation, fibrosis, and mucin secretion. The impact of CFTR activity on these variants after CFTR gene complementation or exposure to CFTR modulators was assessed by molecular and functional studies. Measurements and Main Results: End-stage CF lungs display a stem cell heterogeneity marked by five predominant variants in addition to the normal lung stem cell, of which three are proinflammatory both at the level of gene expression and their ability to drive neutrophilic inflammation in xenografts in immunodeficient mice. The proinflammatory functions of these three variants were unallayed by genetic or pharmacological restoration of CFTR activity. Conclusions: The emergence of three proinflammatory stem cell variants in CF lungs may contribute to the persistence of lung inflammation in patients with CF with advanced disease undergoing CFTR modulator therapy.

Lung Xenotransplantation.

Although efforts have been made to expand the pool of donor lung allografts for human lung transplantation, a shortage remains. Lung xenotransplantation has been proposed as an alternative approach, but lung xenotransplantation in humans has not yet been reported. In addition, significant biological and ethical barriers will have to be addressed before clinical trials can be undertaken. However, significant progress has been made toward addressing biological incompatibilities that present a barrier, and recent advances in genetic engineering tools promise to accelerate further progress.

Orthotopic Ferret Tracheal Transplantation Using a Recellularized Bioengineered Graft Produces Functional Epithelia.

Tracheal grafts may be necessary to bridge long-segment defects after curative resection for airway obstructions. Bioengineered grafts have emerged as an appealing option, given the possibilities of altering the histologic and cellular profile of the conduit. We previously designed a bioreactor capable of luminally decellularizing and recellularizing a ferret trachea with surface airway epithelia (SAE) basal cells (BCs), and we sought to assess the fate of these grafts when transplanted in an orthotopic fashion. As adjuncts to the procedure, we investigated the use of a vascular endothelial growth factor (VEGF)-laden hydrogel and of immunosuppression (IS) in graft revascularization and viability. IS was shown to limit early graft revascularization, but this effect could be counteracted with VEGF supplementation. Submucosal gland (SMG) loss was shown to be inevitable regardless of the revascularization strategy. Lastly, the bioengineered tracheas survived one month after transplant with differentiation of our implanted BCs that then transitioned into a recipient-derived functional epithelium. The work presented in this manuscript has important implications for future cellular and regenerative therapies.

Development and characterization of ferret ex vivo tracheal injury and cell engraftment model.

The field of airway biology research relies primarily on in vitro and in vivo models of disease and injury. The use of ex vivo models to study airway injury and cell-based therapies remains largely unexplored although such models have the potential to overcome certain limitations of working with live animals and may more closely replicate in vivo processes than in vitro models can. Here, we characterized a ferret ex vivo tracheal injury and cell engraftment model. We describe a protocol for whole-mount staining of cleared tracheal explants, and showed that it provides a more comprehensive structural overview of the surface airway epithelium (SAE) and submucosal glands (SMGs) than 2D sections, revealing previously underappreciated structural anatomy of tracheal innervation and vascularization. Using an ex vivo model of tracheal injury, we evaluated the injury responses in the SAE and SMGs that turned out to be consistent with published in vivo work. We used this model to assess factors that influence engraftment of transgenic cells, providing a system for optimizing cell-based therapies. Finally, we developed a novel 3D-printed reusable culture chamber that enables live imaging of tracheal explants and differentiation of engrafted cells at an air-liquid interface. These approaches promise to be useful for modeling pulmonary diseases and testing therapies. Graphical abstract1,2. We describe here a method for differential mechanical injury of ferret tracheal explants that can be used to evaluate airway injury responses ex vivo. 3. Injured explants can be cultured at ALI (using the novel tissue-transwell device on the right) and submerged long-term to evaluate tissue-autonomous regeneration responses. 4. Tracheal explants can also be used for low throughput screens of compounds to improve cell engraftment efficiency or can be seeded with particular cells to model a disease phenotype. 5. Lastly, we demonstrate that ex vivo-cultured tracheal explants can be evaluated by various molecular assays and by immunofluorescent imaging that can be performed live using our custom-designed tissue-transwell.

Cloning a profibrotic stem cell variant in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, and rapidly fatal interstitial lung disease marked by the replacement of lung alveoli with dense fibrotic matrices. Although the mechanisms initiating IPF remain unclear, rare and common alleles of genes expressed in lung epithelia, combined with aging, contribute to the risk for this condition. Consistently, single-cell RNA sequencing (scRNA-seq) studies have identified lung basal cell heterogeneity in IPF that might be pathogenic. We used single-cell cloning technologies to generate "libraries" of basal stem cells from the distal lungs of 16 patients with IPF and 10 controls. We identified a major stem cell variant that was distinguished from normal stem cells by its ability to transform normal lung fibroblasts into pathogenic myofibroblasts in vitro and to activate and recruit myofibroblasts in clonal xenografts. This profibrotic stem cell variant, which was shown to preexist in low quantities in normal and even fetal lungs, expressed a broad network of genes implicated in organ fibrosis and showed overlap in gene expression with abnormal epithelial signatures identified in previously published scRNA-seq studies of IPF. Drug screens highlighted specific vulnerabilities of this profibrotic variant to inhibitors of epidermal growth factor and mammalian target of rapamycin signaling as prospective therapeutic targets. This profibrotic stem cell variant in IPF was distinct from recently identified profibrotic stem cell variants in chronic obstructive pulmonary disease and may extend the notion that inappropriate accrual of minor and preexisting stem cell variants contributes to chronic lung conditions.

Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells.

Keratin expression dynamically changes in airway basal cells (BCs) after acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In bronchiolitis obliterans (BO) after lung transplantation, BC clonogenicity declines, which is associated with a switch from keratin15 (Krt15) to keratin14 (Krt14). We investigated these keratins' roles using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs did not differentiate into club and ciliated cells but had enhanced clonogenicity. By contrast, Krt15-KO did not alter BC differentiation but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo after injury. Krt14, but not Krt15, bound the tumor suppressor stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, whereas dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts with Krt14-KO phenotype and resembles the phenotype in BO with decreased clonogenicity, increased Krt14, and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior, which is relevant in chronic disease states like BO.

A Matched Survival Analysis of Lung Transplant Recipients With Coronavirus Disease 2019-Related Respiratory Failure.

BACKGROUND: Lung transplantation is an acceptable and potentially life-saving treatment option for coronavirus disease 2019 (COVID-19)-induced acute respiratory distress syndrome and pulmonary fibrosis. This study was conducted to determine whether recipients of lung transplantation (LT) for COVID-19-related lung disease have comparable outcomes to other recipients with a similar level of lung dysfunction. METHODS: The Organ Procurement and Transplant Network database was queried for adult LT candidates between 2006 and 2021. Recipients with COVID-19-related respiratory failure were matched 1:2 using a nearest-neighbor algorithm. Kaplan-Meier methods with log-rank tests were used to compare long-term survival. A proportional hazards model was used to calculate risk of death. RESULTS: A total of 37,333 LT candidates from all causes were compared with 334 candidates from COVID-19-related respiratory failure. COVID-19 recipients were more likely to be younger (50 vs 57 years, P < .001), male (79% vs 60%, P < .001), require extracorporeal membrane oxygenation (56.3% vs 4.0%, P < .001), and have worse lung function (lung allocation score, 82.4 vs 47.8; P < .001) at transplantation. Subsequently, 227 COVID-19 recipients were matched with 454 controls. Patients who received a transplant for COVID-19 had similar rates of mechanical ventilation, extracorporeal membrane oxygenation, postoperative complications, and functional status at discharge compared with controls. There was no difference in overall survival or risk of death from COVID-19 (hazard ratio, 0.82; 95% CI, 0.45-1.53; P = .54). CONCLUSIONS: Six-month survival for recipients of LT for COVID-19-related respiratory failure was comparable to that of other LT recipients.

Sox9 and Lef1 Regulate the Fate and Behavior of Airway Glandular Progenitors in Response to Injury.

Cartilaginous airways of larger mammals and the mouse trachea contain at least 3 well-established stem cell compartments, including basal cells of the surface airway epithelium (SAE) and ductal and myoepithelial cells of the submucosal glands (SMG). Here we demonstrate that glandular Sox9-expressing progenitors capable of SAE repair decline with age in mice. Notably, Sox9-lineage glandular progenitors produced basal and ciliated cells in the SAE, but failed to produce secretory cells. Lef1 was required for glandular Sox9 lineage contribution to SAE repair, and its deletion significantly reduced proliferation following injury. By contrast, in vivo deletion of Sox9 enhanced proliferation of progenitors in both the SAE and SMG shortly following injury, but these progenitors failed to proliferate in vitro in the absence of Sox9, similar to that previously shown for Lef1 deletion. In cystic fibrosis ferret airways, Sox9 expression inversely correlated with Ki67 proliferative marker expression in SMG and the SAE. Using in vitro and ex vivo models, we demonstrate that Sox9 is extinguished as glandular progenitors exit ducts and proliferate on the airway surface and that Sox9 is required for migration and proper differentiation of SMG, but not surface airway, progenitors. We propose a model whereby Wnt/Lef1 and Sox9 signals differentially regulate the proliferative and migratory behavior of glandular progenitors, respectively.

Ferret Lung Transplantation Models Differential Lymphoid Aggregate Morphology Between Restrictive and Obstructive Forms of Chronic Lung Allograft Dysfunction.

BACKGROUND: Long-term survival after lung transplantation remains limited by chronic lung allograft dysfunction (CLAD). CLAD has 2 histologic phenotypes, namely obliterative bronchiolitis (OB) and restrictive alveolar fibroelastosis (AFE), which have distinct clinical presentations, pathologies, and outcomes. Understanding of OB versus AFE pathogenesis would improve with better animal models. METHODS: We utilized a ferret orthotopic single-lung transplantation model to characterize allograft fibrosis as a histologic measure of CLAD. Native lobes and "No CLAD" allografts lacking aberrant histology were used as controls. We used morphometric analysis to evaluate the size and abundance of B-cell aggregates and tertiary lymphoid organs (TLOs) and their cell composition. Quantitative RNA expression of 47 target genes was performed simultaneously using a custom QuantiGene Plex Assay. RESULTS: Ferret lung allografts develop the full spectrum of human CLAD histology including OB and AFE subtypes. While both OB and AFE allografts developed TLOs, TLO size and number were greater with AFE histology. More activated germinal center cells marked by B-cell lymphoma 6 Transcription Repressor, (B-cell lymphoma 6) expression and fewer cells expressing forkhead box P3 correlated with AFE, congruent with greater diffuse immunoglobulin, plasma cell abundance, and complement 4d staining. Furthermore, forkhead box P3 RNA induction was significant in OB allografts specifically. RNA expression changes were seen in native lobes of animals with AFE but not OB when compared with No CLAD native lobes. CONCLUSIONS: The orthotopic ferret single-lung transplant model provides unique opportunities to better understand factors that dispose allografts to OB versus AFE. This will help develop potential immunomodulatory therapies and antifibrotic approaches for lung transplant patients.

A Novel Bioreactor for Reconstitution of the Epithelium and Submucosal Glands in Decellularized Ferret Tracheas.

Tracheal grafts introduce the possibility to treat airway pathologies that require resection. While there has been success with engraftment of the surface airway epithelium (SAE) onto decellularized tracheas, there has been minimal advancement in regenerating the submucosal glands (SMGs). We designed a cost-effective open-system perfusion bioreactor to investigate the engraftment potential of ferret SAEs and murine myoepithelial cells (MECs) on a partly decellularized ferret trachea with the goal of creating a fully functional tracheal replacement. An air-liquid interface was also arranged by perfusing humidified air through the lumen of a recellularized conduit to induce differentiation. Our versatile bioreactor design was shown to support the successful partial decellularization and recellularization of ferret tracheas. The decellularized grafts maintained biomechanical integrity and chondrocyte viability, consistent with other publications. The scaffolds supported SAE basal cell engraftment, and early differentiation was observed once an air-liquid interface had been established. Lastly, MEC engraftment was sustained, with evidence of diffuse SMG reconstitution. This model will help shed light on SMG regeneration and basal cell differentiation in vitro for the development of fully functional tracheal grafts before transplantation.

Challenges, highlights, and opportunities in cellular transplantation: A white paper of the current landscape.

Although cellular transplantation remains a relatively small field compared to solid organ transplantation, the prospects for advancement in basic science and clinical care remain bountiful. In this review, notable historical events and the current landscape of the field of cellular transplantation are reviewed with an emphasis on islets (allo- and xeno-), hepatocytes (including bioartificial liver), adoptive regulatory immunotherapy, and stem cells (SCs, specifically endogenous organ-specific and mesenchymal). Also, the nascent but rapidly evolving field of three-dimensional bioprinting is highlighted, including its major processing steps and latest achievements. To reach its full potential where cellular transplants are a more viable alternative than solid organ transplants, fundamental change in how the field is regulated and advanced is needed. Greater public and private investment in the development of cellular transplantation is required. Furthermore, consistent with the call of multiple national transplant societies for allo-islet transplants, the oversight of cellular transplants should mirror that of solid organ transplants and not be classified under the unsustainable, outdated model that requires licensing as a drug with the Food and Drug Administration. Cellular transplantation has the potential to bring profound benefit through progress in bioengineering and regenerative medicine, limiting immunosuppression-related toxicity, and providing markedly reduced surgical morbidity.

Impact of paravertebral blocks on analgesic and non-analgesic outcomes after video-assisted thoracoscopic surgery: A propensity matched cohort study.

BACKGROUND: Regional analgesic techniques such as paravertebral blocks (PVBs) have been popularized for analgesia following video-assisted thoracoscopic surgery (VATS). In this single center retrospective propensity matched cohort of subjects, we investigate the impact of paravertebral blocks on the analgesic and non-analgesic outcomes. METHODS: Institutional database was queried to identify all patients undergoing VATS between January 2013 and July 2019 and these patients were divided into those who received paravertebral blocks in combination with general anesthesia (GA) [PVB group] and those who received GA without paravertebral blocks [GA group]. Propensity score matching based on common patient confounders were used to identify patients in each group. Primary outcomes of the study were average pain scores and opioid consumption in the first 24 hours. Secondary analgesic outcomes included pain scores and opioid requirements at other timepoints over the first 48 hours. Non analgesic outcomes were obtained from STS General Thoracic Surgery Database and included length of hospital stay, need for ICU admission, composite outcome of any complication during the hospital course and 30-day mortality. Exploratory analyses were conducted to investigate the impact of PVB on analgesia following different types of surgery and as to whether any other covariates had a greater influence on the included patient centered outcomes. MAIN RESULTS: After propensity score matching, a total of 520 patients (260 per group) were selected for the study out of 1095 patients. The opioid consumption in terms of oral morphine milligram equivalent (MME) [Median (IQR)] for the first 24 hours was significantly lower with the use of PVB [PVB group- 78.5 (96.75); GA group-127.0 (111.5); p<0.001] while the average pain scores in the first 24 hours did not differ significantly [PVB group-4.71 (2.28); GA group-4.85 (2.30); p = 0.70]. The length of hospital stay, opioid requirements at other timepoints, need for ICU admission in the immediate post-operative period and the composite outcome-'any complication' (35% vs 48%) were significantly lower with the use of PVB. Subgroup analysis showed a longer duration of benefit following major lung surgeries compared to others. CONCLUSION: Paravertebral blocks reduced the length of stay and opioid consumption up to 48 hours after VATS without significantly impacting pain scores.

The Role of Definitive Radiation and Surgery in Metastatic Esophageal Cancer: An NCDB Investigation.

BACKGROUND: Approximately 40% of patients with esophageal cancer present with metastatic disease. Survival with palliative treatment is poor, and the benefit of aggressive focal therapies is unclear. This study aimed to identify a subset of patients with metastatic esophageal cancer with favorable outcomes after curative doses of radiation therapy, esophagectomy, or both. METHODS: Between 2004 and 2015, the study investigators found 28,101 patients with metastatic esophageal cancer in the National Cancer Database and identified those who underwent chemotherapy and definitive radiation therapy with or without surgery over the study period. The study compared the estimated median overall survival (OS) of all patients with metastatic esophageal cancer with the estimated median OS of patients with metastatic esophageal cancer who underwent radiation therapy with or without surgery. Multivariable analysis was used to examine clinical and pathologic factors associated with OS. RESULTS: At a median follow-up of 11.1 months, 3219 patients with a median age of 64 years and a radiation dose of 50.4 Gy were identified. Only 202 (6.2%) patients undergoing definitive-dose radiation therapy underwent esophagectomy, with a median age of 60 years. The median OS durations for all patients, for patients treated with radiation, and for patients treated with radiation therapy in combination with esophagectomy were 6.6, 11.5, and 30.2 months, respectively. Among patients undergoing surgery, median OS after surgery was 23.7 months. Patients with lung, liver, or bone metastases were less likely to undergo esophagectomy. On multivariable analysis, esophagectomy and low tumor grade were associated with higher OS, whereas liver and bone metastases at diagnosis were associated with worse OS. CONCLUSIONS: This analysis suggests that select subsets of patients with primarily nonvisceral, nonosseous metastatic esophageal cancer have favorable survival and may potentially benefit from aggressive local therapies.

Safety and Efficacy of Stereotactic Body Radiation Therapy for Locoregional Recurrences After Prior Chemoradiation for Advanced Esophageal Carcinoma.

Purpose: This study aimed to investigate the feasibility of stereotactic body radiation therapy (SBRT) as salvage therapy for locally recurrent esophageal cancer. We hypothesized that SBRT would provide durable treated tumor control with minimal associated toxicity in patients with progressive disease after definitive radiation, chemotherapy, and surgical resection. Methods: This single-institution retrospective study assessed outcomes in patients who received SBRT for locoregional failure of esophageal cancer after initial curative-intent treatment. Only patients who had received neoadjuvant chemoradiation (≥41.4 Gy) for esophageal cancer were selected. Subsequent surgical resection was optional but institutional follow-up by an oncologist was required. The primary endpoints of this study were gastrointestinal and constitutional toxicity, scored with the Common Terminology Criteria for Adverse Events v5.0. A secondary outcome, treated-tumor control, was assessed with RECIST v1.1. Results: Nine patients (11 locoregional recurrences) treated with SBRT were reviewed, with a median follow-up time of 10.5 months. Most patients initially presented with T3 (88.9%), N1 (55.6%), moderately differentiated (66.7%) adenocarcinoma (88.9%), and had received a median 50.4 Gy delivered over 28 fractions with concurrent carboplatin/paclitaxel chemotherapy followed by surgical resection. Median time to recurrence was 16.3 months. Median total dose delivered by SBRT was 27.5 Gy (delivered in five fractions). Two patients experienced acute grade 1 fatigue and vomiting. No patient experienced grade 3 or higher toxicity. One patient experienced failure in the SBRT treatment field at 5.8 months after treatment and six patients developed distant failure. The median progression-free survival time for SBRT-treated tumors was 5.0 months, and median overall survival time was 12.9 months. Conclusions: This single-institution study demonstrated the feasibility of SBRT for locoregional recurrence of esophageal cancer with minimal treatment-related toxicity and high rates of treated tumor control. Prospective studies identifying ideal salvage SBRT candidates for locoregional failure as well as validating its safety are needed.

Stem cells and lung regeneration.

The ability to replace defective cells in an airway with cells that can engraft, integrate, and restore a functional epithelium could potentially cure a number of lung diseases. Progress toward the development of strategies to regenerate the adult lung by either in vivo or ex vivo targeting of endogenous stem cells or pluripotent stem cell derivatives is limited by our fundamental lack of understanding of the mechanisms controlling human lung development, the precise identity and function of human lung stem and progenitor cell types, and the genetic and epigenetic control of human lung fate. In this review, we intend to discuss the known stem/progenitor cell populations, their relative differences between rodents and humans, their roles in chronic lung disease, and their therapeutic prospects. Additionally, we highlight the recent breakthroughs that have increased our understanding of these cell types. These advancements include novel lineage-traced animal models and single-cell RNA sequencing of human airway cells, which have provided critical information on the stem cell subtypes, transition states, identifying cell markers, and intricate pathways that commit a stem cell to differentiate or to maintain plasticity. As our capacity to model the human lung evolves, so will our understanding of lung regeneration and our ability to target endogenous stem cells as a therapeutic approach for lung disease.

Regenerative Metaplastic Clones in COPD Lung Drive Inflammation and Fibrosis.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema that represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

Derivation of induced pluripotent stem cells from ferret somatic cells.

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.