The molecular mechanisms of extracellular matrix-derived hydrogel therapy in idiopathic pulmonary fibrosis models.

Idiopathic Pulmonary Fibrosis (IPF) is a progressively debilitating lung condition characterized by oxidative stress, cell phenotype shifts, and excessive extracellular matrix (ECM) deposition. Recent studies have shown promising results using decellularized ECM-derived hydrogels produced through pepsin digestion in various lung injury models and even a human clinical trial for myocardial infarction. This study aimed to characterize the composition of ECM-derived hydrogels, assess their potential to prevent fibrosis in bleomycin-induced IPF models, and unravel their underlying molecular mechanisms of action. Porcine lungs were decellularized and pepsin-digested for 48 h. The hydrogel production process, including visualization of protein molecular weight distribution and hydrogel gelation, was characterized. Peptidomics analysis of ECM-derived hydrogel contained peptides from 224 proteins. Probable bioactive and cell-penetrating peptides, including collagen IV, laminin beta 2, and actin alpha 1, were identified. ECM-derived hydrogel treatment was administered as an early intervention to prevent fibrosis advancement in rat models of bleomycin-induced pulmonary fibrosis. ECM-derived hydrogel concentrations of 1 mg/mL and 2 mg/mL showed subtle but noticeable effects on reducing lung inflammation, oxidative damage, and protein markers related to fibrosis (e.g., alpha-smooth muscle actin, collagen I). Moreover, distinct changes were observed in macroscopic appearance, alveolar structure, collagen deposition, and protein expression between lungs that received ECM-derived hydrogel and control fibrotic lungs. Proteomic analyses revealed significant protein and gene expression changes related to cellular processes, pathways, and components involved in tissue remodeling, inflammation, and cytoskeleton regulation. RNA sequencing highlighted differentially expressed genes associated with various cellular processes, such as tissue remodeling, hormone secretion, cell chemotaxis, and cytoskeleton engagement. This study suggests that ECM-derived hydrogel treatment influence pathways associated with tissue repair, inflammation regulation, cytoskeleton reorganization, and cellular response to injury, potentially offering therapeutic benefits in preventing or mitigating lung fibrosis.

Mechanical control of innate immune responses against viral infection revealed in a human lung alveolus chip.

Mechanical breathing motions have a fundamental function in lung development and disease, but little is known about how they contribute to host innate immunity. Here we use a human lung alveolus chip that experiences cyclic breathing-like deformations to investigate whether physical forces influence innate immune responses to viral infection. Influenza H3N2 infection of mechanically active chips induces a cascade of host responses including increased lung permeability, apoptosis, cell regeneration, cytokines production, and recruitment of circulating immune cells. Comparison with static chips reveals that breathing motions suppress viral replication by activating protective innate immune responses in epithelial and endothelial cells, which are mediated in part through activation of the mechanosensitive ion channel TRPV4 and signaling via receptor for advanced glycation end products (RAGE). RAGE inhibitors suppress cytokines induction, while TRPV4 inhibition attenuates both inflammation and viral burden, in infected chips with breathing motions. Therefore, TRPV4 and RAGE may serve as new targets for therapeutic intervention in patients infected with influenza and other potential pandemic viruses that cause life-threatening lung inflammation.

Orthotopic Transplantation of Human Bioartificial Lung Grafts in a Porcine Model: A Feasibility Study.

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.

Protective Effects of Extracellular Matrix-Derived Hydrogels in Idiopathic Pulmonary Fibrosis.

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.

Modeling pulmonary cystic fibrosis in a human lung airway-on-a-chip.

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.

Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Disease 2019.

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.

AAV-mediated gene therapy targeting TRPV4 mechanotransduction for inhibition of pulmonary vascular leakage.

Enhanced vascular permeability in the lungs can lead to pulmonary edema, impaired gas exchange, and ultimately respiratory failure. While oxygen delivery, mechanical ventilation, and pressure-reducing medications help alleviate these symptoms, they do not treat the underlying disease. Mechanical activation of transient receptor potential vanilloid 4 (TRPV4) ion channels contributes to the development of pulmonary vascular disease, and overexpression of the high homology (HH) domain of the TRPV4-associated transmembrane protein CD98 has been shown to inhibit this pathway. Here, we describe the development of an adeno-associated virus (AAV) vector encoding the CD98 HH domain in which the AAV serotypes and promoters have been optimized for efficient and specific delivery to pulmonary cells. AAV-mediated gene delivery of the CD98 HH domain inhibited TRPV4 mechanotransduction in a specific manner and protected against pulmonary vascular leakage in a human lung Alveolus-on-a-Chip model. As AAV has been used clinically to deliver other gene therapies, these data raise the possibility of using this type of targeted approach to develop mechanotherapeutics that target the TRPV4 pathway for treatment of pulmonary edema in the future.

Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases 2017. An Official American Thoracic Society Workshop Report.

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.

A Fully Automated High-Throughput Bioreactor System for Lung Regeneration.

IMPACT STATEMENT: This work presents methods for ex vivo lung recellularization and biomimetic culture in a high-throughput and consistent manner. These methods allow for the testing of multiple variables, all of which are simultaneously controlled and monitored on a single fully automated pump system, and subsequent assessment of both epithelial and endothelial repair and tissue regeneration. This system provides a controlled environment for tissue repair, wherein key variables can be modified, monitored, reproduced, and optimized to advance the goal of ex vivo tissue regeneration based on native organ scaffolds.

Acellular human lung scaffolds to model lung disease and tissue regeneration.

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.

Bioengineering Human Lung Grafts on Porcine Matrix.

OBJECTIVE: Bioengineering of viable, functional, and implantable human lung grafts on porcine matrix. SUMMARY BACKGROUND DATA: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Bioengineering of functional human induced pluripotent stem cell-derived intestinal grafts.

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.

Regenerative potential of human airway stem cells in lung epithelial engineering.

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.

An Official American Thoracic Society Workshop Report 2015. Stem Cells and Cell Therapies in Lung Biology and Diseases.

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.

Bioengineering Lungs for Transplantation.

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.

Bioengineering Human Myocardium on Native Extracellular Matrix.

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.

Engineering pulmonary vasculature in decellularized rat and human lungs.

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.

Assessment of Proliferation and Cytotoxicity in a Biomimetic Three-Dimensional Model of Lung Cancer.

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.

Using nature's platform to engineer bio-artificial lungs.

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.

Enhanced lung epithelial specification of human induced pluripotent stem cells on decellularized lung matrix.

BACKGROUND: Whole-lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluated the capacity of acellular lung scaffolds to support recellularization with lung progenitors derived from human induced pluripotent stem cells (iPSCs). METHODS: Whole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% sodium dodecyl sulfate solution. Resulting lung scaffolds were cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm, anteriorized to a foregut fate, and then ventralized to a population expressing NK2 homeobox 1 (Nkx2.1). Cells were seeded onto slices and whole lungs, which were maintained under constant perfusion biomimetic culture. Lineage specification was assessed by quantitative polymerase chain reaction and immunofluorescent staining. Regenerated left lungs were transplanted in an orthotopic position. RESULTS: Activin-A treatment, followed by transforming growth factor-ß inhibition, induced differentiation of human iPSCs to anterior foregut endoderm as confirmed by forkhead box protein A2 (FOXA2), SRY (Sex Determining Region Y)-Box 17 (SOX17), and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after 5 days. Cells expressing Nkx2.1 were identified at 40% to 60% efficiency. Within whole-lung scaffolds and under perfusion culture, cells further upregulated Nkx2.1 expression. After orthotopic transplantation, grafts were perfused and ventilated by host vasculature and airways. CONCLUSIONS: Decellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole-organ scaffolds and biomimetic culture enable coseeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further in vivo graft maturation.