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Background Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which results in impaired airway mucociliary clearance, inflammation, infection, and respiratory insufficiency. The development of new therapeutics for CF are limited by the lack of reliable preclinical models that recapitulate the structural, immunological, and bioelectrical features of human CF lungs. Methods We leveraged organ-on-a-chip technology to develop a microfluidic device lined by primary human CF bronchial epithelial cells grown under an air-liquid interface and interfaced with pulmonary microvascular endothelial cells (CF Airway Chip) exposed to fluid flow. The responses of CF and healthy Airway Chips were analyzed in the presence or absence of polymorphonuclear leukocytes (PMNs) and the bacterial pathogen, Pseudomonas aeruginosa. Results The CF Airway Chip faithfully recapitulated many features of the human CF airways, including enhanced mucus accumulation, increased cilia density, and a higher ciliary beating frequency compared to chips lined by healthy bronchial epithelial cells. The CF chips also secreted higher levels of IL-8, which was accompanied by enhanced PMN adhesion to the endothelium and transmigration into the airway compartment. In addition, CF Airway Chips provided a more favorable environment for Pseudomonas aeruginosa growth, which resulted in enhanced secretion of inflammatory cytokines and recruitment of PMNs to the airway. Conclusions The human CF Airway Chip may provide a valuable preclinical tool for pathophysiology studies as well as for drug testing and personalized medicine. more...

Current reconstruction methods of the laryngotracheal segment fail to replace the complex functions of the human larynx. Bioengineering approaches to reconstruction have been limited by the complex tissue compartmentation of the larynx. We attempted to overcome this limitation by bioengineering laryngeal grafts from decellularized canine laryngeal scaffolds recellularized with human primary cells under one uniform culture medium condition. First, we developed laryngeal scaffolds which were generated by detergent perfusion-decellularization over 9 days and preserved their glycosaminoglycan content and biomechanical properties of a native larynx. After subcutaneous implantations in rats for 14 days, the scaffolds did not elicit a CD3 lymphocyte response. We then developed a uniform culture medium that strengthened the endothelial barrier over 5 days after an initial growth phase. Simultaneously, this culture medium supported airway epithelial cell and skeletal myoblast growth while maintaining their full differentiation and maturation potential. We then applied the uniform culture medium composition to whole laryngeal scaffolds seeded with endothelial cells from both carotid arteries and external jugular veins and generated reendothelialized arterial and venous vascular beds. Under the same culture medium, we bioengineered epithelial monolayers onto laryngeal mucosa and repopulated intrinsic laryngeal muscle. We were then able to demonstrate early muscle formation in an intramuscular transplantation model in immunodeficient mice. We supported formation of three humanized laryngeal tissue compartments under one uniform culture condition, possibly a key factor in developing complex, multicellular, ready-to-transplant tissue grafts. Impact Statement For patients undergoing laryngectomy, no reconstruction methods are available to restore the complex functions of the human larynx. The first promising preclinical results have been achieved with the use of biological scaffolds fabricated from decellularized tissue. However, the complexity of laryngeal tissue composition remains a hurdle to create functional viable grafts, since previously each cell type requires tailored culture conditions. In this study, we report the de novo formation of three humanized laryngeal tissue compartments under one uniform culture condition, a possible keystone in creating vital composite tissue grafts for laryngeal regeneration. more...

The University of Vermont Larner College of Medicine, in collaboration with the National Heart, Lung, and Blood Institute (NHLBI), the Alpha-1 Foundation, the American Thoracic Society, the Cystic Fibrosis Foundation, the European Respiratory Society, the International Society for Cell & Gene Therapy, and the Pulmonary Fibrosis Foundation, convened a workshop titled “Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases” from July 24 through 27, 2017, at the University of Vermont, Burlington, Vermont. The conference objectives were to review and discuss current understanding of the following topics: 1) stem and progenitor cell biology and the role that they play in endogenous repair or as cell therapies after lung injury, 2) the emerging role of extracellular vesicles as potential therapies, 3) ex vivo bioengineering of lung and airway tissue, and 4) progress in induced pluripotent stem cell protocols for deriving lung cell types and applications in disease modeling. All of these topics are research areas in which significant and exciting progress has been made over the past few years. In addition, issues surrounding the ethics and regulation of cell therapies worldwide were discussed, with a special emphasis on combating the growing problem of unproven cell interventions being administered to patients with lung diseases. Finally, future research directions were discussed, and opportunities for both basic and translational research were identified. more...

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with significant gas exchange impairment owing to exaggerated extracellular matrix (ECM) deposition and myofibroblast activation. IPF has no cure, and although nintedanib and pirfenidone are two approved medications for symptom management, the total treatment cost is exuberant and prohibitive to a global uninsured patient population. New therapeutic alternatives with moderate costs are needed to treat IPF. ECM hydrogels derived from decellularized lungs are cost-effective therapeutic candidates to treat pulmonary fibrosis because of their reported antioxidant properties. Oxidative stress contributes to IPF pathophysiology by damaging macromolecules, interfering with tissue remodeling, and contributing to myofibroblast activation. Thus, preventing oxidative stress has beneficial outcomes in IPF. For this purpose, this review describes ECM hydrogel's properties to regulate oxidative stress and tissue remodeling in IPF. Impact statement Idiopathic pulmonary fibrosis (IPF) is a disease without a cure and with limited treatment options. At present, approved medications are expensive and pose a huge socioeconomic challenge to patients who depend on them. Affordability and effectiveness are desirable qualities for new therapeutic alternatives. Extracellular matrix hydrogels have properties that distinguish them other biomaterials, and it has been studied in the context of fibrosis-related molecular mechanisms. This review examines the biological processes involved in IPF and suggests developing a hydrogel-based treatment option for patients with IPF. more...

Lung transplantation is the only treatment for end-stage lung disease; however, donor organ shortage and intense immunosuppression limit its broad clinical impact. Bioengineering of lungs with patient-derived cells could overcome these problems. We created bioartificial lungs by seeding human-derived cells onto porcine lung matrices and performed orthotopic transplantation to assess feasibility and in vivo function. Porcine decellularized lung scaffolds were seeded with human airway epithelial cells and human umbilical vein endothelial cells. Following in vitro culture, the bioartificial lungs were orthotopically transplanted into porcine recipients with planned 1-day survival (n = 3). Lungs were assessed with histology and in vivo function. Orthotopic transplantation of cadaveric lungs was performed as control. Engraftment of endothelial and epithelial cells in the grafts were histologically demonstrated. Technically successful orthotopic anastomoses of the vasculatures and airway were achieved in all animals. Perfusion and ventilation of the lung grafts were confirmed intraoperatively. The gas exchange function was evident immediately after transplantation; PO2 gradient between pulmonary artery and vein were 178 ± 153 mm Hg in the bioartificial lung group and 183 ± 117 mm Hg in the control group. At time of evaluation 24 hours after reperfusion, the pulmonary arteries were found to be occluded with thrombus in all bioartificial lungs. Engineering and orthotopic transplantation of bioartificial lungs with human cells were technically feasible in a porcine model. Early gas exchange function was evident. Further progress in optimizing recellularization and maturation of the grafts will be necessary for sustained perfusability and function. more...

A workshop entitled “Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases” was hosted by the University of Vermont Larner College of Medicine in collaboration with the National Heart, Lung and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, the International Society for Cell and Gene Therapy and the Pulmonary Fibrosis Foundation. The event was held from July 15 to 18, 2019 at the University of Vermont, Burlington, Vermont. The objectives of the conference were to review and discuss the current status of the following active areas of research: 1) technological advancements in the analysis and visualisation of lung stem and progenitor cells; 2) evaluation of lung stem and progenitor cells in the context of their interactions with the niche; 3) progress toward the application and delivery of stem and progenitor cells for the treatment of lung diseases such as cystic fibrosis; 4) progress in induced pluripotent stem cell models and application for disease modelling; and 5) the emerging roles of cell therapy and extracellular vesicles in immunomodulation of the lung. This selection of topics represents some of the most dynamic research areas in which incredible progress continues to be made. The workshop also included active discussion on the regulation and commercialisation of regenerative medicine products and concluded with an open discussion to set priorities and recommendations for future research directions in basic and translation lung biology., This workshop report discusses recent advances in cell therapy and bioengineering approaches for repair and regeneration of diseased lungs https://bit.ly/2DqA8eu more...

Patients with short bowel syndrome lack sufficient functional intestine to sustain themselves with enteral intake alone. Transplantable vascularized bioengineered intestine could restore nutrient absorption. Here we report the engineering of humanized intestinal grafts by repopulating decellularized rat intestinal matrix with human induced pluripotent stem cell-derived intestinal epithelium and human endothelium. After 28 days of in vitro culture, hiPSC-derived progenitor cells differentiate into a monolayer of polarized intestinal epithelium. Human endothelial cells seeded via native vasculature restore perfusability. Ex vivo isolated perfusion testing confirms transfer of glucose and medium-chain fatty acids from lumen to venous effluent. Four weeks after transplantation to RNU rats, grafts show survival and maturation of regenerated epithelium. Systemic venous sampling and positron emission tomography confirm uptake of glucose and fatty acids in vivo. Bioengineering intestine on vascularized native scaffolds could bridge the gap between cell/tissue-scale regeneration and whole organ-scale technology needed to treat intestinal failure patients., There is a need for humanised grafts to treat patients with intestinal failure. Here, the authors generate intestinal grafts by recellularizing native intestinal matrix with human induced pluripotent stem cell-derived epithelium and human endothelium, and show nutrient absorption after transplantation in rats. more...

Background: Lung transplantation remains the only treatment for many end-stage lung diseases. However, donor organ shortage and the need for immunosuppression limit clinical impact. Cadaveric lungs can be decellularized to yield scaffolds that provide a suitable platform for tissue regeneration. Directed differentiation of human iPSCs toward a lung epithelial fate is a promising approach to facilitate the goal of whole organ regeneration with patient-specific cells. Objective: To investigate the capacity of iPSC-derived alveolar cells to form distal pulmonary epithelium on acellular lung scaffolds. Methods: Human iPSCs carrying Nkx2.1-GFP and SPC-TdTomato reporters were differentiated towards a lung alveolar fate by established protocols. SP-C+ alveolar cells were delivered to decellularized rat lungs (n=6) to repopulate the distal niche. Epithelial tissue formation was induced through extended ex vivo biomimetic culture. Regenerated lungs were analyzed for tissue architecture and cell fate. Acellular human lung slices were also repopulated with iPSC-derived cells in vitro. Seeded lung slices (n=12) were cultured, then tissue structure and cell fate were also analyzed. Results: Formation of early distal lung epithelial tissue with mature type II alveolar cell phenotype, expressing Nkx2.1, E-Cadherin, SP-C, SP-B was observed. We also identified AQP5 positive cells, which may identify a type 1 alveolar fate. Similar findings were seen on human lung slices. Conclusions: Human iPSC-derived alveolar epithelial cells can form distal lung epithelium on both rodent and human lung scaffolds, demonstrating the future potential for patient-specific lung regeneration at a transplantable scale. more...

Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure, without the risk of rejection. Building upon the process of whole organ perfusion decellularization, we aimed to develop novel, translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5+TP63+ basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation, in combination with primary pulmonary endothelial cells. To show clinical scalability, and to test the regenerative capacity of the basal cell population in a human context, we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology, and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair. more...

Whole lung extracellular matrix scaffolds can be created by perfusion of cadaveric organs with decellularizing detergents, providing a platform for organ regeneration. Lung epithelial engineering must address both the proximal airway cells that function to metabolize toxins and aid mucociliary clearance and the distal pneumocytes that facilitate gas exchange. Engineered pulmonary vasculature must support in vivo blood perfusion with low resistance and intact barrier function and be antithrombotic. Repopulating the native lung matrix with sufficient cell numbers in appropriate anatomic locations is required to enable organ function. more...

Recent advances in whole lung bioengineering have opened new doors for studying lung repair and regeneration ex vivo using acellular human derived lung tissue scaffolds. Methods to decellularise whole human lungs, lobes or resected segments from normal and diseased human lungs have been developed using both perfusion and immersion based techniques. Immersion based techniques allow laboratories without access to intact lobes the ability to generate acellular human lung scaffolds. Acellular human lung scaffolds can be further processed into small segments, thin slices or extracellular matrix extracts, to study cell behaviour such as viability, proliferation, migration and differentiation. Recent studies have offered important proof of concept of generating sufficient primary endothelial and lung epithelial cells to recellularise whole lobes that can be maintained for several days ex vivo in a bioreactor to study regeneration. In parallel, acellular human lung scaffolds have been increasingly used for studying cell–extracellular environment interactions. These studies have helped provide new insights into the role of the matrix and the extracellular environment in chronic human lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Acellular human lung scaffolds are a versatile new tool for studying human lung repair and regeneration ex vivo. more...

Rationale: More than 25 million individuals have heart failure worldwide, with ≈4000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only ≈2500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. Objective: The objective of this study is to translate previous work to human scale and clinically relevant cells for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human induced pluripotent stem cell–derived cardiomyocytes. Methods and Results: To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiomyocytes derived from nontransgenic human induced pluripotent stem cells and generated tissues of increasing 3-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole-heart scaffolds with human induced pluripotent stem cell–derived cardiomyocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue and showed electrical conductivity, left ventricular pressure development, and metabolic function. Conclusions: Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human induced pluripotent stem cell–derived cardiomyocytes and enable the bioengineering of functional human myocardial-like tissue of multiple complexities. more...

Bioengineered lungs produced from patient-derived cells may one day provide an alternative to donor lungs for transplantation therapy. Here we report the regeneration of functional pulmonary vasculature by repopulating the vascular compartment of decellularized rat and human lung scaffolds with human cells, including endothelial and perivascular cells derived from induced pluripotent stem cells. We describe improved methods for delivering cells into the lung scaffold and for maturing newly formed endothelium through co-seeding of endothelial and perivascular cells and a two-phase culture protocol. Using these methods we achieved ∼75% endothelial coverage in the rat lung scaffold relative to that of native lung. The regenerated endothelium showed reduced vascular resistance and improved barrier function over the course of in vitro culture and remained patent for 3 days after orthotopic transplantation in rats. Finally, we scaled our approach to the human lung lobe and achieved efficient cell delivery, maintenance of cell viability and establishment of perfusable vascular lumens. more...

Approximately 100,000 individuals in the United States currently await kidney transplantation, and 400,000 individuals live with end-stage kidney disease requiring hemodialysis. The creation of a transplantable graft to permanently replace kidney function would address donor organ shortage and the morbidity associated with immunosuppression. Such a bioengineered graft must have the kidney's architecture and function and permit perfusion, filtration, secretion, absorption and drainage of urine. We decellularized rat, porcine and human kidneys by detergent perfusion, yielding acellular scaffolds with vascular, cortical and medullary architecture, a collecting system and ureters. To regenerate functional tissue, we seeded rat kidney scaffolds with epithelial and endothelial cells and perfused these cell-seeded constructs in a whole-organ bioreactor. The resulting grafts produced rudimentary urine in vitro when perfused through their intrinsic vascular bed. When transplanted in an orthotopic position in rat, the grafts were perfused by the recipient's circulation and produced urine through the ureteral conduit in vivo. more...

Bioengineering of viable, functional, and implantable human lung grafts on porcine matrix.Implantable bioartificial organ grafts could revolutionize transplant surgery. To date, several milestones toward that goal have been achieved in rodent models. To make bioengineered organ grafts clinically relevant, scaling to human cells and graft size are the next steps.We seeded porcine decellularized lung scaffolds with human airway epithelial progenitor cells derived from rejected donor lungs, and banked human umbilical vein endothelial cells. We subsequently enabled tissue formation in whole organ culture. The resulting grafts were then either analyzed in vitro (n = 15) or transplanted into porcine recipients in vivo (n = 3).By repopulating porcine extracellular matrix scaffolds with human endothelial cells, we generated pulmonary vasculature with mature endothelial lining and sufficient anti-thrombotic function to enable blood perfusion. By repopulating the epithelial surface with human epithelial progenitor cells, we created a living, functioning gas exchange graft. After surgical implantation, the bioengineered lung grafts were able to withstand physiological blood flow from the recipient's pulmonary circulation, and exchanged gases upon ventilation during the 1-hour observation.Engineering and transplantation of viable lung grafts based on decellularized porcine lung scaffolds and human endothelial and epithelial cells is technically feasible. Further graft maturation will be necessary to enable higher-level functions such as mucociliary clearance, and ventilation-perfusion matching. more...

The University of Vermont College of Medicine, in collaboration with the NHLBI, Alpha-1 Foundation, American Thoracic Society, Cystic Fibrosis Foundation, European Respiratory Society, International Society for Cellular Therapy, and the Pulmonary Fibrosis Foundation, convened a workshop, "Stem Cells and Cell Therapies in Lung Biology and Lung Diseases," held July 27 to 30, 2015, at the University of Vermont. The conference objectives were to review the current understanding of the role of stem and progenitor cells in lung repair after injury and to review the current status of cell therapy and ex vivo bioengineering approaches for lung diseases. These are all rapidly expanding areas of study that both provide further insight into and challenge traditional views of mechanisms of lung repair after injury and pathogenesis of several lung diseases. The goals of the conference were to summarize the current state of the field, discuss and debate current controversies, and identify future research directions and opportunities for both basic and translational research in cell-based therapies for lung diseases. This 10th anniversary conference was a follow up to five previous biennial conferences held at the University of Vermont in 2005, 2007, 2009, 2011, and 2013. Each of those conferences, also sponsored by the National Institutes of Health, American Thoracic Society, and respiratory disease foundations, has been important in helping guide research and funding priorities. The major conference recommendations are summarized at the end of the report and highlight both the significant progress and major challenges in these rapidly progressing fields. more...

Native lung extracellular matrix can be isolated from cadaveric organs via perfusion decellularization and provides a novel scaffold material for lung engineering. Based on this platform, several proof-of-principle studies have demonstrated the feasibility of whole organ recellularization and culture in rodent models and have helped us better understand the numerous challenges in up-scaling to clinically relevant tissues. Standardized protocols to generate whole lung scaffolds of porcine and human scale have been reported, but our understanding of the remaining extracellular matrix components and their properties is incomplete. Effective recellularization will require the isolation and in vitro expansion of clinically relevant cell sources, either from primary or stem cell-derived populations, and techniques to effectively deliver these populations throughout the lung scaffold. Ultimately, only tightly controlled recapitulation of tissue development and repair in vitro will enable us to mature lung grafts to function before implantation. Although substantial progress has been made, we are only beginning to grasp the complexity of this exciting new technology. more...

Usual interstitial pneumonia (UIP) is characterized by extracellular matrix deposition and the development of pulmonary fibrosis. Fibroblastic foci found in the lung are believed to represent an early stage in the evolution of this disease.To compare gene expression profiles in different components of lung tissue (fibroblastic foci, adjacent epithelium, and areas of type 2 pneumocyte hyperplasia) from patients with UIP, and contrast these profiles to distal, uninvolved (control) alveolar tissue from patients undergoing lung resection for cancer.Lung resection tissue (UIP, n = 11; controls, n = 11) was snap-frozen for subsequent laser capture microdissection, followed by mRNA extraction, linear amplification, and quantitative real-time polymerase chain reaction.In patients with UIP, tissue inhibitor of matrix metalloprotease-1 and matrix metalloprotease (MMP)-2 gene expression was up-regulated within the fibroblastic foci compared with the overlying epithelium (p = 0.03, p = 0.02), and to control alveoli (p = 0.001, p = 0.04), respectively. MMP-9 and MMP-7, as well as osteopontin, were up-regulated in fibroblastic foci (p = 0.01, p = 0.08, p = 0.08), the adjacent epithelium (p = 0.001, p = 0.001, p = 0.03), and the hyperplastic type 2 pneumocytes (p = 0.02, p = 0.001, p = 0.08), respectively, compared with control alveoli.Altered gene expression of important profibrotic mediators in the different cellular lung compartments in patients with UIP likely plays an important role in pathogenesis of the deranged extracellular matrix deposition and subsequent fibrosis in this condition. more...

Background Decellularized whole-organ scaffolds show great potential in cancer research. They have been used in the biomimetic three-dimensional (3D) culture of non-small cell lung cancer cells, allowing the study of unique aspects of lung cancer biology. However, there are no reproducible assays capable of directly monitoring processes involved in cancer progression within such scaffolds. Methods The human adenocarcinoma cell lines H358, PC9, and SW1573 were subjected to biomimetic 3D culture within decellularized lung scaffolds. A resazurin-based reagent was perfused through the scaffold to determine cell viability over the culture period and in response to treatment with cisplatin or erlotinib. Results The resazurin reduction perfusion assay detected a progressive increase in the reduction of resazurin over time for all cell lines cultured within decellularized lung scaffolds, translating into incremental cell populations. Also, it detected a positive cytotoxic effect in H358- and PC9-seeded scaffolds after treatment with cisplatin, and in PC9-seeded scaffolds after treatment with erlotinib. Moreover, it identified relative resistance to erlotinib in H358- and SW1573-seeded scaffolds. Results from this assay correlated with histopathology, expression of caspase 3, and activity of epidermal growth factor receptor signaling. Conclusions The methods described here for the monitoring of lung cancer cell viability under biomimetic 3D culture conditions within decellularized lung scaffolds permit the study of cancer cell proliferation, the evaluation of responses to therapeutic interventions, and the determination of relative chemo-sensitivities. more...

The primary treatment for end-stage lung disease is lung transplantation. However, donor organ shortage remains a major barrier for many patients. In recent years, techniques for maintaining lungs ex vivo for evaluation and short-term ( more...

Background Chronic lung diseases are marked by progressive inflammation, tissue damage and remodelling. Bone marrow-derived progenitor cells may contribute to these processes. The objectives of this study were to (1) to quantify CD45+Collagen-1+ fibrocytes and a novel epithelial-like population of bone marrow-derived cells, which express Clara Cell Secretory Protein, in patients at the time of lung transplant and (2) to evaluate mediators that may act to recruit these cells during injury. Methods Using an observational design, progenitor cells were quantified by flow cytometry from both bone marrow (BM) and peripheral blood (PB). Migration was tested using in vitro transwell assays. Multiplex bead-based assays were used to quantify plasma cytokines. Results An increase in CD45+Collagen-1+ fibrocytes was found in pulmonary fibrosis and bronchiolitis obliterans patients. Cystic fibrosis patients had an increase in CCSP+ cells in both the BM and PB. The proportion of CCSP+ cells in the BM and PB was correlated. CCSP+ cells express the chemokine receptors CCR2, CCR4, CXCR3, and CXCR4, and significantly migrated in vitro toward Stromal Derived Factor-1 (SDF-1) and Stem Cell Growth Factor-β (SCGF-β). Plasma cytokine levels differed between disease groups, with a significant correlation between SCGF-β and CCSP+ cells and between Monocyte Chemotactic Protein-1 and fibrocytes. Conclusions Different bone marrow-derived cells are found in various lung diseases. Increased fibrocytes were associated with fibrotic lung diseases. An increase in the novel CCSP+ epithelial-like progenitors in cystic fibrosis patients was found. These differences may be mediated by alterations in plasma cytokines responsible for cell recruitment. more...

The clinical management of idiopathic pulmonary fibrosis (IPF) remains a major challenge due to lack of effective drug therapy or accurate indicators for disease progression. Fibrocytes are circulating mesenchymal cell progenitors that are involved in tissue repair and fibrosis.To test the hypothesis that assay of these cells may provide a biomarker for activity and progression of IPF.Fibrocytes were defined as cells positive for CD45 and collagen-1 by flow cytometry and quantified in patients with stable IPF and during acute exacerbation of the disease. We investigated the clinical and prognostic value of fibrocyte counts by comparison with standard clinical parameters and survival. We used healthy age-matched volunteers and patients with acute respiratory distress syndrome as control subjects.Fibrocytes were significantly elevated in patients with stable IPF (n = 51), with a further increase during acute disease exacerbation (n = 7; P0.001 vs. control subjects). Patients with acute respiratory distress syndrome (n = 10) were not different from healthy control subjects or stable patients with IPF. Fibrocyte numbers were not correlated with lung function or radiologic severity scores, but they were an independent predictor of early mortality. The mean survival of patients with fibrocytes higher than 5% of total blood leukocytes was 7.5 months compared with 27 months for patients with less than 5% (P0.0001).Fibrocytes are an indicator for disease activity of IPF and might be useful as a clinical marker for disease progression. This study suggests that quantification of circulating fibrocytes may allow prediction of early mortality in patients with IPF. more...

Purpose Organ engineering is a theoretical therapy for organ failure. Whole organ matrices can be created by detergent perfusion, generating acellular scaffolds with the capacity for recellularization. This study aimed to up-scale this technology to clinically relevant porcine and human lungs, and to test their recellularization potential in biomimetic culture. Methods and Materials Rat, pig, and human lungs were perfused with detergent via the pulmonary artery at constant pressures, followed by washing. DNA was quantified by PicoGreen. Residual SDS was quantified by Stains-All assay. Soluble collagen, elastin, and glycosaminoglycans were quantified by colourimetric assays. Proteomic analysis by mass spectrometry (LC-MS/MS) generated spectral counts as a measure of abundance. Perfusion recellularization experiments of human lungs used standard organ culture techniques derived from rodent protocols. Results Rat lungs decellularized using (1) Sodium Dodecyl Sulfate (SDS), (2) Sodium deoxycholate, or (3) CHAPS showed loss of DNA but greatest preservation of ECM in SDS decellularized lungs. Histology and proteomics also confirmed greater matrix preservation in SDS lungs. The SDS-based decellularization protocol was up-scaled to porcine (n=7) and human (n=3) lungs with greater SDS concentration and perfusion pressures, and extended decellularization time. Removal of residual DNA and SDS was confirmed. Analysis of 10 areas from each decellularized lung showed intact ECM and lung architecture. Proteomics of human decellularized lungs further demonstrated matrix preservation. Recellularization of isolated lobar scaffolds used endothelial (HUVEC) and lung epithelial (SAEC) lines delivered to the appropriate organ compartment. Throughout biomimetic organ culture matrix integrity was maintained, while cell retention was observed by H&E and DAPI. Conclusions SDS decellularization can generate organ scaffolds with preserved ECM and architecture in whole porcine and human lungs. The results confirm clinical scale lung scaffolds can support recellularization for organ regeneration. more...

Introduction: Viral oncolysis has been proposed as an adjuvant therapy for the treatment of advanced lung cancer. A more complex model would allow for improved in vitro testing of this therapy prior to moving towards clinical application. Methods: Lung blocks were harvested from Sprague Dawley rats and subjected to perfusion-decellularization via the pulmonary artery with 0.1% sodium dodecyl sulfate and 1% Triton X-100 to obtain decellularized whole-lung scaffolds. Decellularized lung scaffolds were placed in a bioreactor to allow perfusion of cell-specific media through the pulmonary artery. Human lung cancer cells were seeded into the scaffolds by tracheal delivery of cell suspensions containing 20-30 million cells. After 7-10 days of culture in the bioreactor, lung scaffolds harboring tumor nodules were treated with a replication-conditional Herpes Simplex Virus type-1 (hrR3) at a dose of 1x108 Plaque Forming Units delivered through the pulmonary artery. In parallel, scaffolds with tumors treated with cisplatin served as positive controls while delivery of phosphate-buffered saline served as a negative control. Tissue was collected 48 hours after treatment delivery and was analyzed by histology, immunostaining for Lac Z to allow the visualization and localization of replicating virus, and by assessment of cytotoxicity with a formazan-based assay. Results: There were no residual cellular elements after the decellularization process as demonstrated by histologic analysis, and the scaffolds showed preserved microarchitecture of the alveolar spaces and vasculature. Human lung cancer cell lines H358 and SW1573 showed engraftment and tumor nodule formation and growth inside the decellularized lung scaffolds. The custom-designed bioreactor system allowed for delivery of treatments including hrR3 through the pulmonary artery, in an easy and convenient way while maintaining sterility. Treatment with hrR3 decreased the total tumor burden per mg of tissue to 92% of that seen in negative controls in SW1573-seeded scaffolds and to 52% in H358-seeded scaffolds. Both SW1573- and H358-seeded scaffolds treated with cisplatin showed an overall >99% decrease in tumor burden. Tumors treated with hrR3 showed different morphology when compared to negative controls. Conclusions: Decellularized lung scaffolds can serve as a more realistic three-dimensional model for the in vitro study of viral oncolysis of human lung cancer, as they account for anatomical variations and provide natural micro-architectural barriers to oncolytic virus such as basement membranes. Differences in the efficacy of viral oncolysis according to epithelial vs. mesenchymal tumor phenotypes should be further investigated. Citation Format: Luis F. Tapias, Sarah E. Gilpin, Justin Elliott, Roshini Zachariah, Haiyu Zhou, Bryan C. Fuchs, Lan Wei, Danielle K. Deperalta, Kumudu D. Kuruppu, Kenneth K. Tanabe, Harald C. Ott, Michael Lanuti. A 3D model for lung cancer based on decellularized lung scaffolds allows for in vitro testing of viral oncolysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2037. doi:10.1158/1538-7445.AM2014-2037 more...