Organ Boundary Circuits Regulate Sox9+ Alveolar Tuft Cells During Post-Pneumonectomy Lung Regeneration.

Tissue homeostasis is controlled by cellular circuits governing cell growth, organization, and differentation. In this study we identify previously undescribed cell-to-cell communication that mediates information flow from mechanosensitive pleural mesothelial cells to alveolar-resident stem-like tuft cells in the lung. We find mesothelial cells to express a combination of mechanotransduction genes and lineage-restricted ligands which makes them uniquely capable of responding to tissue tension and producing paracrine cues acting on parenchymal populations. In parallel, we describe a large population of stem-like alveolar tuft cells that express the endodermal stem cell markers Sox9 and Lgr5 and a receptor profile making them uniquely sensitive to cues produced by pleural Mesothelium. We hypothesized that crosstalk from mesothelial cells to alveolar tuft cells might be central to the regulation of post-penumonectomy lung regeneration. Following pneumonectomy, we find that mesothelial cells display radically altered phenotype and ligand expression, in a pattern that closely tracks with parenchymal epithelial proliferation and alveolar tissue growth. During an initial pro-inflammatory stage of tissue regeneration, Mesothelium promotes epithelial proliferation via WNT ligand secretion, orchestrates an increase in microvascular permeability, and encourages immune extravasation via chemokine secretion. This stage is followed first by a tissue remodeling period, characterized by angiogenesis and BMP pathway sensitization, and then a stable return to homeostasis. Coupled with key changes in parenchymal structure and matrix production, the cumulative effect is a now larger organ including newly-grown, fully-functional tissue parenchyma. This study paints Mesothelial cells as a key orchestrating cell type that defines the boundary of the lung and exerts critical influence over the tissue-level signaling state regulating resident stem cell populations. The cellular circuits unearthed here suggest that human lung regeneration might be inducible through well-engineered approaches targeting the induction of tissue regeneration and safe return to homeostasis.

Peripheral Blood Single-Cell Sequencing Uncovers Common and Specific Immune Aberrations in Fibrotic Lung Diseases.

Rationale and Objectives: The extent and commonality of peripheral blood immune aberrations in fibrotic interstitial lung diseases are not well characterized. In this study, we aimed to identify common and distinct immune aberrations in patients with idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (FHP) using cutting-edge single-cell profiling technologies. Methods: Single-cell RNA sequencing was performed on patients and healthy controls' peripheral blood and bronchoalveolar lavage samples using 10X Genomics 5' gene expression and V(D)J profiling. Cell type composition, transcriptional profiles, cellular trajectories and signaling, and T and B cell receptor repertoires were studied. The standard Seurat R pipeline was followed for cell type composition and differential gene expression analyses. Transcription factor activity was imputed using the DoRothEA-VIPER algorithm. Pseudotime analyses were conducted using Monocle3, while RNA velocity analyses were performed with Velocyto, scVelo, and CellRank. Cell-cell connectomics were assessed using the Connectome R package. V(D)J analyses were conducted using CellRanger and Immcantation frameworks. Across all analyses, disease group differences were assessed using the Wilcoxon rank-sum test. Measurements and Main Results: 327,990 cells from 83 samples were profiled. Overall, changes in monocytes were common to IPF and FHP, whereas lymphocytes exhibited disease-specific aberrations. Both diseases displayed enrichment of CCL3 hi /CCL4 hi CD14+ monocytes (p<2.2e-16) and S100A hi CD14+ monocytes (p<2.2e-16) versus controls. Trajectory and RNA velocity analysis suggested that pro-fibrotic macrophages observed in BAL originated from peripheral blood monocytes. Lymphocytes exhibited disease-specific aberrations, with CD8+ GZMK hi T cells and activated B cells primarily enriched in FHP patients. V(D)J analyses revealed unique T and B cell receptor complementarity-determining region 3 (CDR3) amino acid compositions (p<0.05) in FHP and significant IgA enrichment in IPF (p<5.2e-7). Conclusions: We identified common and disease-specific immune mechanisms in IPF and FHP; S100A hi monocytes and SPP1 hi macrophages are common to IPF and FHP, whereas GMZK hi T lymphocytes and T and B cell receptor repertoires were unique in FHP. Our findings open novel strategies for the diagnosis and treatment of IPF and FHP.

Rational engineering of lung alveolar epithelium.

Engineered whole lungs may one day expand therapeutic options for patients with end-stage lung disease. However, the feasibility of ex vivo lung regeneration remains limited by the inability to recapitulate mature, functional alveolar epithelium. Here, we modulate multimodal components of the alveolar epithelial type 2 cell (AEC2) niche in decellularized lung scaffolds in order to guide AEC2 behavior for epithelial regeneration. First, endothelial cells coordinate with fibroblasts, in the presence of soluble growth and maturation factors, to promote alveolar scaffold population with surfactant-secreting AEC2s. Subsequent withdrawal of Wnt and FGF agonism synergizes with tidal-magnitude mechanical strain to induce the differentiation of AEC2s to squamous type 1 AECs (AEC1s) in cultured alveoli, in situ. These results outline a rational strategy to engineer an epithelium of AEC2s and AEC1s contained within epithelial-mesenchymal-endothelial alveolar-like units, and highlight the critical interplay amongst cellular, biochemical, and mechanical niche cues within the reconstituting alveolus.

SARS-CoV-2 leverages airway epithelial protective mechanism for viral infection.

Despite much concerted effort to better understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection, relatively little is known about the dynamics of early viral entry and infection in the airway. Here we analyzed a single-cell RNA sequencing dataset of early SARS-CoV-2 infection in a humanized in vitro model, to elucidate key mechanisms by which the virus triggers a cell-systems-level response in the bronchial epithelium. We find that SARS-CoV-2 virus preferentially enters the tissue via ciliated cell precursors, giving rise to a population of infected mature ciliated cells, which signal to basal cells, inducing further rapid differentiation. This feedforward loop of infection is mitigated by further cell-cell communication, before interferon signaling begins at three days post-infection. These findings suggest hijacking by the virus of potentially beneficial tissue repair mechanisms, possibly exacerbating the outcome. This work both elucidates the interplay between barrier tissues and viral infections and may suggest alternative therapeutic approaches targeting non-immune response mechanisms.

Comprehensive visualization of cell-cell interactions in single-cell and spatial transcriptomics with NICHES.

MOTIVATION: Recent years have seen the release of several toolsets that reveal cell-cell interactions from single-cell data. However, all existing approaches leverage mean celltype gene expression values, and do not preserve the single-cell fidelity of the original data. Here, we present NICHES (Niche Interactions and Communication Heterogeneity in Extracellular Signaling), a tool to explore extracellular signaling at the truly single-cell level. RESULTS: NICHES allows embedding of ligand-receptor signal proxies to visualize heterogeneous signaling archetypes within cell clusters, between cell clusters and across experimental conditions. When applied to spatial transcriptomic data, NICHES can be used to reflect local cellular microenvironment. NICHES can operate with any list of ligand-receptor signaling mechanisms, is compatible with existing single-cell packages, and allows rapid, flexible analysis of cell-cell signaling at single-cell resolution. AVAILABILITY AND IMPLEMENTATION: NICHES is an open-source software implemented in R under academic free license v3.0 and it is available at http://github.com/msraredon/NICHES. Use-case vignettes are available at https://msraredon.github.io/NICHES/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Fibroblast inflammatory priming determines regenerative versus fibrotic skin repair in reindeer.

Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.

Computation and visualization of cell-cell signaling topologies in single-cell systems data using Connectome.

Single-cell RNA-sequencing data has revolutionized our ability to understand of the patterns of cell-cell and ligand-receptor connectivity that influence the function of tissues and organs. However, the quantification and visualization of these patterns in a way that informs tissue biology are major computational and epistemological challenges. Here, we present Connectome, a software package for R which facilitates rapid calculation and interactive exploration of cell-cell signaling network topologies contained in single-cell RNA-sequencing data. Connectome can be used with any reference set of known ligand-receptor mechanisms. It has built-in functionality to facilitate differential and comparative connectomics, in which signaling networks are compared between tissue systems. Connectome focuses on computational and graphical tools designed to analyze and explore cell-cell connectivity patterns across disparate single-cell datasets and reveal biologic insight. We present approaches to quantify focused network topologies and discuss some of the biologic theory leading to their design.

Single-cell multi-omics reveals dyssynchrony of the innate and adaptive immune system in progressive COVID-19.

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100Ahi/HLA-DRlo classical monocytes and activated LAG-3hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8+ clones, unmutated IGHG+ B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19.

Characterization of the COPD alveolar niche using single-cell RNA sequencing.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide, however our understanding of cell specific mechanisms underlying COPD pathobiology remains incomplete. Here, we analyze single-cell RNA sequencing profiles of explanted lung tissue from subjects with advanced COPD or control lungs, and we validate findings using single-cell RNA sequencing of lungs from mice exposed to 10 months of cigarette smoke, RNA sequencing of isolated human alveolar epithelial cells, functional in vitro models, and in situ hybridization and immunostaining of human lung tissue samples. We identify a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance in COPD. Using transcriptomic network analyses, we predict capillary endothelial cells are inflamed in COPD, particularly through increased CXCL-motif chemokine signaling. Finally, we detect a high-metallothionein expressing macrophage subpopulation enriched in advanced COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD.

Pressure-Regulated Ventilator Splitting for Disaster Relief: Design, Testing, and Clinical Experience.

The coronavirus disease 2019 (COVID-19) pandemic has revealed that even the best-resourced hospitals may lack sufficient ventilators to support patients under surge conditions. During a pandemic or mass trauma, an affordable, low-maintenance, off-the-shelf device that would allow health care teams to rapidly expand their ventilator capacity could prove lifesaving, but only if it can be safely integrated into a complex and rapidly changing clinical environment. Here, we define an approach to safe ventilator sharing that prioritizes predictable and independent care of patients sharing a ventilator. Subsequently, we detail the design and testing of a ventilator-splitting circuit that follows this approach and describe our clinical experience with this circuit during the COVID-19 pandemic. This circuit was able to provide individualized and titratable ventilatory support with individualized positive end-expiratory pressure (PEEP) to 2 critically ill patients at the same time, while insulating each patient from changes in the other's condition. We share insights from our experience using this technology in the intensive care unit and outline recommendations for future clinical applications.

A Pulmonary Vascular Model From Endothelialized Whole Organ Scaffolds.

The development of an in vitro system for the study of lung vascular disease is critical to understanding human pathologies. Conventional culture systems fail to fully recapitulate native microenvironmental conditions and are typically limited in their ability to represent human pathophysiology for the study of disease and drug mechanisms. Whole organ decellularization provides a means to developing a construct that recapitulates structural, mechanical, and biological features of a complete vascular structure. Here, we developed a culture protocol to improve endothelial cell coverage in whole lung scaffolds and used single-cell RNA-sequencing analysis to explore the impact of decellularized whole lung scaffolds on endothelial phenotypes and functions in a biomimetic bioreactor system. Intriguingly, we found that the phenotype and functional signals of primary pulmonary microvascular revert back-at least partially-toward native lung endothelium. Additionally, human induced pluripotent stem cell-derived endothelium cultured in decellularized lung systems start to gain various native human endothelial phenotypes. Vascular barrier function was partially restored, while small capillaries remained patent in endothelial cell-repopulated lungs. To evaluate the ability of the engineered endothelium to modulate permeability in response to exogenous stimuli, lipopolysaccharide (LPS) was introduced into repopulated lungs to simulate acute lung injury. After LPS treatment, proinflammatory signals were significantly increased and the vascular barrier was impaired. Taken together, these results demonstrate a novel platform that recapitulates some pulmonary microvascular functions and phenotypes at a whole organ level. This development may help pave the way for using the whole organ engineering approach to model vascular diseases.

Mechanisms of Hypoxia-Induced Pulmonary Arterial Stiffening in Mice Revealed by a Functional Genetics Assay of Structural, Functional, and Transcriptomic Data.

Hypoxia adversely affects the pulmonary circulation of mammals, including vasoconstriction leading to elevated pulmonary arterial pressures. The clinical importance of changes in the structure and function of the large, elastic pulmonary arteries is gaining increased attention, particularly regarding impact in multiple chronic cardiopulmonary conditions. We establish a multi-disciplinary workflow to understand better transcriptional, microstructural, and functional changes of the pulmonary artery in response to sustained hypoxia and how these changes inter-relate. We exposed adult male C57BL/6J mice to normoxic or hypoxic (FiO2 10%) conditions. Excised pulmonary arteries were profiled transcriptionally using single cell RNA sequencing, imaged with multiphoton microscopy to determine microstructural features under in vivo relevant multiaxial loading, and phenotyped biomechanically to quantify associated changes in material stiffness and vasoactive capacity. Pulmonary arteries of hypoxic mice exhibited an increased material stiffness that was likely due to collagen remodeling rather than excessive deposition (fibrosis), a change in smooth muscle cell phenotype reflected by decreased contractility and altered orientation aligning these cells in the same direction as the remodeled collagen fibers, endothelial proliferation likely representing endothelial-to-mesenchymal transitioning, and a network of cell-type specific transcriptomic changes that drove these changes. These many changes resulted in a system-level increase in pulmonary arterial pulse wave velocity, which may drive a positive feedback loop exacerbating all changes. These findings demonstrate the power of a multi-scale genetic-functional assay. They also highlight the need for systems-level analyses to determine which of the many changes are clinically significant and may be potential therapeutic targets.

Microvascular fluid flow in ex vivo and engineered lungs.

In recent years, it has become common to experiment with ex vivo perfused lungs for organ transplantation and to attempt regenerative pulmonary engineering using decellularized lung matrices. However, our understanding of the physiology of ex vivo organ perfusion is imperfect; it is not currently well understood how decreasing microvascular barrier affects the perfusion of pulmonary parenchyma. In addition, protocols for lung perfusion and organ culture fluid-handling are far from standardized, with widespread variation on both basic methods and on ideally controlled parameters. To address both of these deficits, a robust, noninvasive, and mechanistic model is needed which is able to predict microvascular resistance and permeability in perfused lungs while providing insight into capillary recruitment. Although validated mathematical models exist for fluid flow in native pulmonary tissue, previous models generally assume minimal intravascular leak from artery to vein and do not assess capillary bed recruitment. Such models are difficult to apply to both ex vivo lung perfusions, in which edema can develop over time and microvessels can become blocked, and to decellularized ex vivo organomimetic cultures, in which microvascular recruitment is variable and arterially perfused fluid enters into the alveolar space. Here, we develop a mathematical model of pulmonary microvascular fluid flow which is applicable in both instances, and we apply our model to data from native, decellularized, and regenerating lungs under ex vivo perfusion. The results provide substantial insight into microvascular pressure-flow mechanics, while producing previously unknown output values for tissue-specific capillary-alveolar hydraulic conductivity, microvascular recruitment, and total organ barrier resistance.NEW & NOTEWORTHY We present a validated model of pulmonary microvascular fluid mechanics and apply this model to study the effects of increased capillary permeability in decellularized and regenerating lungs. We find that decellularization alters microvascular steady-state mechanics and that re-endothelialization partially rescues key biologic parameters. The described model provides powerful insight into intraorgan microvascular dynamics and may be used to guide regenerative engineering experiments. We include all data and derivations necessary to replicate this work.

High resolution stereolithography fabrication of perfusable scaffolds to enable long-term meso-scale hepatic culture for disease modeling.

Microphysiological systems (MPS), comprising human cell cultured in formats that capture features of the three-dimensional (3D) microenvironments of native human organs under microperfusion, are promising tools for biomedical research. Here we report the development of a mesoscale physiological system (MePS) enabling the long-term 3D perfused culture of primary human hepatocytes at scales of over 106cells per MPS. A central feature of the MePS, which employs a commercially-available multiwell bioreactor for perfusion, is a novel scaffold comprising a dense network of nano- and micro-porous polymer channels, designed to provide appropriate convective and diffusive mass transfer of oxygen and other nutrients while maintaining physiological values of shear stress. The scaffold design is realized by a high resolution stereolithography fabrication process employing a novel resin. This new culture system sustains mesoscopic hepatic tissue-like cultures with greater hepatic functionality (assessed by albumin and urea synthesis, and CYP3A4 activity) and lower inflammation markers compared to comparable cultures on the commercial polystyrene scaffold. To illustrate applications to disease modeling, we established an insulin-resistant phenotype by exposing liver cells to hyperglycemic and hyperinsulinemic media. Future applications of the MePS include the co-culture of hepatocytes with resident immune cells and the integration with multiple organs to model complex liver-associated diseases.

Integrated Single-Cell Atlas of Endothelial Cells of the Human Lung.

BACKGROUND: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium. METHODS: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension. RESULTS: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1, and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic-venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). CONCLUSIONS: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

Bioengineering the Blood-gas Barrier.

The pulmonary blood-gas barrier represents a remarkable feat of engineering. It achieves the exquisite thinness needed for gas exchange by diffusion, the strength to withstand the stresses and strains of repetitive and changing ventilation, and the ability to actively maintain itself under varied demands. Understanding the design principles of this barrier is essential to understanding a variety of lung diseases, and to successfully regenerating or artificially recapitulating the barrier ex vivo. Many classical studies helped to elucidate the unique structure and morphology of the mammalian blood-gas barrier, and ongoing investigations have helped to refine these descriptions and to understand the biological aspects of blood-gas barrier function and regulation. This article reviews the key features of the blood-gas barrier that enable achievement of the necessary design criteria and describes the mechanical environment to which the barrier is exposed. It then focuses on the biological and mechanical components of the barrier that preserve integrity during homeostasis, but which may be compromised in certain pathophysiological states, leading to disease. Finally, this article summarizes recent key advances in efforts to engineer the blood-gas barrier ex vivo, using the platforms of lung-on-a-chip and tissue-engineered whole lungs. © 2020 American Physiological Society. Compr Physiol 10:415-452, 2020.

Platform Effects on Regeneration by Pulmonary Basal Cells as Evaluated by Single-Cell RNA Sequencing.

Cell-based therapies have shown promise for treating myriad chronic pulmonary diseases through direct application of epithelial progenitors or by way of engineered tissue grafts or whole organs. To elucidate environmental effects on epithelial regenerative outcomes in vitro, here, we isolate and culture a population of pharmacologically expanded basal cells (peBCs) from rat tracheas. At peak basal marker expression, we simultaneously split peBCs into four in vitro platforms: organoid, air-liquid interface (ALI), engineered trachea, and engineered lung. Following differentiation, these samples are evaluated using single-cell RNA sequencing (scRNA-seq) and computational pipelines are developed to compare samples both globally and at the population level. A sample of native rat tracheal epithelium is also evaluated by scRNA-seq as a control for engineered epithelium. Overall, this work identifies platform-specific effects that support the use of engineered models to achieve the most physiologic differential outcomes in pulmonary epithelial regenerative applications.

Single-cell connectomic analysis of adult mammalian lungs.

Efforts to decipher chronic lung disease and to reconstitute functional lung tissue through regenerative medicine have been hampered by an incomplete understanding of cell-cell interactions governing tissue homeostasis. Because the structure of mammalian lungs is highly conserved at the histologic level, we hypothesized that there are evolutionarily conserved homeostatic mechanisms that keep the fine architecture of the lung in balance. We have leveraged single-cell RNA sequencing techniques to identify conserved patterns of cell-cell cross-talk in adult mammalian lungs, analyzing mouse, rat, pig, and human pulmonary tissues. Specific stereotyped functional roles for each cell type in the distal lung are observed, with alveolar type I cells having a major role in the regulation of tissue homeostasis. This paper provides a systems-level portrait of signaling between alveolar cell populations. These methods may be applicable to other organs, providing a roadmap for identifying key pathways governing pathophysiology and informing regenerative efforts.

Engineering of human brain organoids with a functional vascular-like system.

Human cortical organoids (hCOs), derived from human embryonic stem cells (hESCs), provide a platform to study human brain development and diseases in complex three-dimensional tissue. However, current hCOs lack microvasculature, resulting in limited oxygen and nutrient delivery to the inner-most parts of hCOs. We engineered hESCs to ectopically express human ETS variant 2 (ETV2). ETV2-expressing cells in hCOs contributed to forming a complex vascular-like network in hCOs. Importantly, the presence of vasculature-like structures resulted in enhanced functional maturation of organoids. We found that vascularized hCOs (vhCOs) acquired several blood-brain barrier characteristics, including an increase in the expression of tight junctions, nutrient transporters and trans-endothelial electrical resistance. Finally, ETV2-induced endothelium supported the formation of perfused blood vessels in vivo. These vhCOs form vasculature-like structures that resemble the vasculature in early prenatal brain, and they present a robust model to study brain disease in vitro.

Non-invasive and real-time measurement of microvascular barrier in intact lungs.

Microvascular leak is a phenomenon witnessed in multiple disease states. In organ engineering, regaining a functional barrier is the most crucial step towards creating an implantable organ. All previous methods of measuring microvascular permeability were either invasive, lengthy, introduced exogenous macromolecules, or relied on extrapolations from cultured cells. We present here a system that enables real-time measurement of microvascular permeability in intact rat lungs. Our unique system design allows direct, non-invasive measurement of average alveolar and capillary pressures, tracks flow paths within the organ, and enables calculation of lumped internal resistances including microvascular barrier. We first describe the physiology of native and decellularized lungs and the inherent properties of the extracellular matrix as functions of perfusion rate. We next track changing internal resistances and flows in injured native rat lungs, resolving the onset of microvascular leak, quantifying changing vascular resistances, and identifying distinct phases of organ failure. Finally, we measure changes in permeability within engineered lungs seeded with microvascular endothelial cells, quantifying cellular effects on internal vascular and barrier resistances over time. This system marks considerable progress in bioreactor design for intact organs and may be used to monitor and garner physiological insights into native, decellularized, and engineered tissues.