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.

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.

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.

Engineered Lung Tissues Prepared from Decellularized Lung Slices.

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 µm-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

Lung Tissue Engineering: Toward a More Deliberate Approach.

Chronic lung disease remains a leading cause of morbidity and mortality. Given the dearth of definitive therapeutic options, there is an urgent need to augment the pool of donor organs for transplantation. One strategy entails building a lung ex vivo in the laboratory. The past decade of whole lung tissue engineering has laid a foundation of systems and strategies for this approach. Meanwhile, tremendous progress in lung stem cell biology is elucidating cues contributing to alveolar repair, and speaks to the potential of whole lung regeneration in the future. This perspective discusses the key challenges facing the field and highlights opportunities to combine insights from biology with engineering strategies to adopt a more deliberate, and ultimately successful, approach to lung engineering.

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.

Development of a Bioartificial Vascular Pancreas.

Transplantation of pancreatic islets has been shown to be effective, in some patients, for the long-term treatment of type 1 diabetes. However, transplantation of islets into either the portal vein or the subcutaneous space can be limited by insufficient oxygen transfer, leading to islet loss. Furthermore, oxygen diffusion limitations can be magnified when islet numbers are increased dramatically, as in translating from rodent studies to human-scale treatments. To address these limitations, an islet transplantation approach using an acellular vascular graft as a vascular scaffold has been developed, termed the BioVascular Pancreas (BVP). To create the BVP, islets are seeded as an outer coating on the surface of an acellular vascular graft, using fibrin as a hydrogel carrier. The BVP can then be anastomosed as an arterial (or arteriovenous) graft, which allows fully oxygenated arterial blood with a pO2 of roughly 100 mmHg to flow through the graft lumen and thereby supply oxygen to the islets. In silico simulations and in vitro bioreactor experiments show that the BVP design provides adequate survivability for islets and helps avoid islet hypoxia. When implanted as end-to-end abdominal aorta grafts in nude rats, BVPs were able to restore near-normoglycemia durably for 90 days and developed robust microvascular infiltration from the host. Furthermore, pilot implantations in pigs were performed, which demonstrated the scalability of the technology. Given the potential benefits provided by the BVP, this tissue design may eventually serve as a solution for transplantation of pancreatic islets to treat or cure type 1 diabetes.

An ex vivo physiologic and hyperplastic vessel culture model to study intra-arterial stent therapies.

Conventional in vitro methods for biological evaluation of intra-arterial devices such as stents fail to accurately predict cytotoxicity and remodeling events. An ex vivo flow-tunable vascular bioreactor system (VesselBRx), comprising intra- and extra-luminal monitoring capabilities, addresses these limitations. VesselBRx mimics the in vivo physiological, hyperplastic, and cytocompatibility events of absorbable magnesium (Mg)-based stents in ex vivo stent-treated porcine and human coronary arteries, with in-situ and real-time monitoring of local stent degradation effects. Unlike conventional, static cell culture, the VesselBRx perfusion system eliminates unphysiologically high intracellular Mg2+ concentrations and localized O2 consumption resulting from stent degradation. Whereas static stented arteries exhibited only 20.1% cell viability and upregulated apoptosis, necrosis, metallic ion, and hypoxia-related gene signatures, stented arteries in VesselBRx showed almost identical cell viability to in vivo rabbit models (~94.0%). Hyperplastic intimal remodeling developed in unstented arteries subjected to low shear stress, but was inhibited by Mg-based stents in VesselBRx, similarly to in vivo. VesselBRx represents a critical advance from the current static culture standard of testing absorbable vascular implants.

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.

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.

Fas ligand and nitric oxide combination to control smooth muscle growth while sparing endothelium.

Metallic stents cause vascular wall damage with subsequent smooth muscle cell (SMC) proliferation, neointimal hyperplasia, and treatment failure. To combat in-stent restenosis, drug-eluting stents (DES) delivering mTOR inhibitors such as sirolimus or everolimus have become standard for coronary stenting. However, the relatively non-specific action of mTOR inhibitors prevents efficient endothelium recovery and mandates dual antiplatelet therapy to prevent thrombosis. Unfortunately, long-term dual antiplatelet therapy leads to increased risk of bleeding/stroke and, paradoxically, myocardial infarction. Here, we took advantage of the fact that nitric oxide (NO) increases Fas receptors on the SMC surface. Fas forms a death-inducing complex upon binding to Fas ligand (FasL), while endothelial cells (ECs) are relatively resistant to this pathway. Selected doses of FasL and NO donor synergistically increased SMC apoptosis and inhibited SMC growth more potently than did everolimus or sirolimus, while having no significant effect on EC viability and proliferation. This differential effect was corroborated in ex vivo pig coronaries, where the neointimal formation was inhibited by the drug combination, but endothelial viability was retained. We also deployed FasL-NO donor-releasing ethylene-vinyl acetate copolymer (EVAc)-coated stents into pig coronary arteries, and cultured them in perfusion bioreactors for one week. FasL and NO donor, released from the stent coating, killed SMCs close to the stent struts, even in the presence of flow rates mimicking those of native arteries. Thus, the FasL-NO donor-combination has a potential to prevent intimal hyperplasia and in-stent restenosis, without harming endothelial restoration, and hence may be a superior drug delivery strategy for DES.

Lung regeneration: steps toward clinical implementation and use.

PURPOSE OF REVIEW: Whole lung tissue engineering is a relatively new area of investigation. In a short time, however, the field has advanced quickly beyond proof of concept studies in rodents and now stands on the cusp of wide-spread scale up to large animal studies. Therefore, this technology is ever closer to being directly clinically relevant. RECENT FINDINGS: The main themes in the literature include refinement of the fundamental components of whole lung engineering and increasing effort to direct induced pluripotent stem cells and lung progenitor cells toward use in lung regeneration. There is also increasing need for and emphasis on functional evaluation in the lab and in vivo, and the use of all of these tools to construct and evaluate forthcoming clinically scaled engineered lung. SUMMARY: Ultimately, the goal of the research described herein is to create a useful clinical product. In the intermediate time, however, the tools described here may be employed to advance our knowledge of lung biology and the organ-specific regenerative capacity of lung stem and progenitor cells.

Targeted proteomics effectively quantifies differences between native lung and detergent-decellularized lung extracellular matrices.

Extracellular matrix is a key component of many products in regenerative medicine. Multiple regenerative medicine products currently in the clinic are comprised of human or xenogeneic extracellular matrix. In addition, whole-organ regeneration exploits decellularized native organs as scaffolds for organotypic cell culture. However, precise understanding of the constituents of such extracellular matrix-based implants and scaffolds has sorely lagged behind their use. We present here an advanced protein extraction method using known quantities of proteotypic 13C-labeled peptides to quantify matrix proteins in native and decellularized lung tissues. Using quantitative proteomics that produce picomole-level measurements of a large number of matrix proteins, we show that a mild decellularization technique ("Triton/SDC") results in near-native retention of laminins, proteoglycans, and other basement membrane and ECM-associated proteins. Retention of these biologically important glycoproteins and proteoglycans is quantified to be up to 27-fold higher in gently-decellularized lung scaffolds compared to scaffolds generated using a previously published decellularization regimen. Cells seeded onto this new decellularized matrix also proliferate robustly, showing positive staining for proliferating cell nuclear antigen (PCNA). The high fidelity of the gently decellularized scaffold as compared to the original lung extracellular matrix represents an important step forward in the ultimate recapitulation of whole organs using tissue-engineering techniques. This method of ECM and scaffold protein analysis allows for better understanding, and ultimately quality control, of matrices that are used for tissue engineering and human implantation. These results should advance regenerative medicine in general, and whole organ regeneration in particular. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) in large part defines the biochemical and mechanical properties of tissues and organs; these inherent cues make acellular ECM scaffolds potent substrates for tissue regeneration. As such, they are increasingly prevalent in the clinic and the laboratory. However, the exact composition of these scaffolds has been difficult to ascertain. This paper uses targeted proteomics to definitively quantify 71 proteins present in acellular lung ECM scaffolds. We use this technique to compare two decellularization methods and demonstrate superior retention of ECM proteins important for cell adhesion, migration, proliferation, and differentiation in scaffolds treated with low-concentration detergent solutions. In the long term, the ability to acquire quantitative biochemical data about biological substrates will facilitate the rational design of engineered tissues and organs based on precise cell-matrix interactions.

Production of decellularized porcine lung scaffolds for use in tissue engineering.

There is a growing body of work dedicated to producing acellular lung scaffolds for use in regenerative medicine by decellularizing donor lungs of various species. These scaffolds typically undergo substantial matrix damage due to the harsh conditions required to remove cellular material (e.g., high pH, strong detergents), lengthy processing times, or pre-existing tissue contamination from microbial colonization. In this work, a new decellularization technique is described that maintains the global tissue architecture, key matrix components, mechanical composition and cell-seeding potential of lung tissue while effectively removing resident cellular material. Acellular lung scaffolds were produced from native porcine lungs using a combination of Triton X-100 and sodium deoxycholate (SDC) at low concentrations in 24 hours. We assessed the effect of matrix decellularization by measuring residual DNA, biochemical composition, mechanical characteristics, tissue architecture, and recellularization capacity.

Fate of distal lung epithelium cultured in a decellularized lung extracellular matrix.

Type II cells are the defenders of the alveolus. They produce surfactant to prevent alveolar collapse, they actively transport water to prevent filling of the air sacs that would otherwise prevent gas exchange, and they differentiate to type I epithelial cells. They are an indispensable component of functional lung tissue. To understand the functionality of type II cells in isolation, we sought to track their fate in decellularized matrices and to assess their ability to contribute to barrier function by differentiation to type I alveolar epithelial cells. Rat type II cells were isolated from neonatal rat lungs by labeling with the RTII-70 surface marker and separation using a magnetic column. This produced a population of ∼50% RTII-70-positive cells accompanied by few type I epithelial cells or α-actin-positive mesenchymal cells. This population was seeded into decellularized rat lung matrices and cultured for 1 or 7 days. Culture in Dulbecco's modified Eagle's medium +10% fetal bovine serum (FBS) resulted in reduced expression of epithelial markers and increased expression of mesenchymal markers. By 7 days, no epithelial markers were visible by immunostaining; nearly all cells were α-actin positive. Gene expression for the mesenchymal markers, α-actin, vimentin, and TGF-ßR, was significantly upregulated on day 1 (p=0.0005, 0.0005, and 2.342E-5, respectively). Transcript levels of α-actin and TGF-ßR remained high at 7 days (p=1.364E-10 and 0.0002). Interestingly, human type II cells cultured under the same conditions showed a similar trend in the loss of epithelial markers, but did not display high expression of mesenchymal markers. Rat cells additionally showed the ability to produce and degrade the basement membrane and extracellular matrix components, such as fibronectin, collagen IV, and collagen I. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) showed significant increases in expression of the fibronectin and matrix metalloprotease-2 (MMP-2) genes after 1 day in culture (p=0.0135 and 0.0128, respectively) and elevated collagen I expression at 7 days (p=0.0016). These data suggest that the original type II-enriched population underwent a transition to increased expression of mesenchymal markers, perhaps as part of a survival or wound-healing program. These results suggest that additional medium components and/or the application of physiologically appropriate stimuli such as ventilation may be required to promote lung-specific epithelial phenotypes.