Stem cells, cell therapies, and bioengineering in lung biology and diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade, increasing evidence from preclinical models suggests that mesenchymal stromal cells, which are not normally resident in the lung, can be used to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathological remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

Hydrogel-Embedded Precision-Cut Lung Slices Model Lung Cancer Premalignancy Ex Vivo.

Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best prevention, 50% of lung cancer diagnoses occur in people who have quit smoking. Research into treatment options for high-risk patients is constrained to rodent models, which are time-consuming, expensive, and require large cohorts. Embedding precision-cut lung slices (PCLS) within an engineered hydrogel and exposing this tissue to vinyl carbamate, a carcinogen from cigarette smoke, creates an in vitro model of lung cancer premalignancy. Hydrogel formulations are selected to promote early lung cancer cellular phenotypes and extend PCLS viability to six weeks. Hydrogel-embedded PCLS are exposed to vinyl carbamate, which induces adenocarcinoma in mice. Analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content after six weeks reveals that vinyl carbamate induces premalignant lesions with a mixed adenoma/squamous phenotype. Putative chemoprevention agents diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue are validated with hydrogel-embedded human PCLS and results show increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the foundation for more sophisticated ex vivo models that enable the study of carcinogenesis and chemoprevention strategies.

Pathogenesis of E-Cigarette Vaping Product Use-Associated Lung Injury (EVALI).

EVALI is an acute inflammatory disease in response to lung cell injury induced by electronic cigarettes and vaping devices (EV) frequently containing Vitamin E Acetate or tetrahydrocannabinol additives, in the context of risk factors such as microbial exposure. EVALI resembles a respiratory viral illness that may progress to acute respiratory failure and acute respiratory distress syndrome (ARDS) but can also affect extra pulmonary organs. Manifestations may be severe, leading to death or long-term morbidity and current treatments are largely supportive. While COVID-19 has demanded public and research attention, EVALI continues to affect young individuals and its better understanding via research remains a priority. Although clinical research led to improved recognition of triggers, clinical and pathological manifestations, and natural course of EVALI, important questions remain that require a better understanding of disease pathogenesis. Preclinical models utilizing laboratory animals and cell or tissue culture platforms provide insight into the physiologic and mechanistic consequences of acute and chronic EV exposure, including the characteristics of the respiratory dysfunction and inflammatory response. However, a key limitation in the field is the absence of an established animal model of EVALI. Important areas of research emphasis include identifying triggers and risk factors to understand why only certain vapers develop EVALI, the role of specific lung immune and structural cells in the pathogenesis of EVALI, and the most important molecular mediators and therapeutic targets in EVALI. © 2023 American Physiological Society. Compr Physiol 13:4617-4630, 2023.

Tissue-engineered models of lung cancer premalignancy.

Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best preventive action, nearly 50% of all lung cancer diagnoses occur in people who have already quit smoking. Research into treatment options for these high-risk patients has been constrained to rodent models of chemical carcinogenesis, which are time-consuming, expensive, and require large numbers of animals. Here we show that embedding precision-cut lung slices within an engineered hydrogel and exposing this tissue to a carcinogen from cigarette smoke creates an in vitro model of lung cancer premalignancy. Hydrogel formulations were selected to promote early lung cancer cellular phenotypes and extend PCLS viability up to six weeks. In this study, hydrogel-embedded lung slices were exposed to the cigarette smoke derived carcinogen vinyl carbamate, which induces adenocarcinoma in mice. At six weeks, analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content revealed that vinyl carbamate induced the formation of premalignant lesions with a mixed adenoma/squamous phenotype. Two putative chemoprevention agents were able to freely diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue were validated with hydrogel-embedded human PCLS and results showed increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the starting point for more sophisticated ex vivo models and a foundation for the study of carcinogenesis and chemoprevention strategies.

Cigarette smoke-induced airspace disease in mice develops independently of HIF-1α signaling in leukocytes.

The pathogenesis of chronic obstructive pulmonary disease (COPD), a prevalent disease primarily caused by cigarette smoke exposure, is incompletely elucidated. Studies in humans and mice have suggested that hypoxia-inducible factor-1α (HIF-1α) may play a role. Reduced lung levels of HIF-1α are associated with decreased vascular density, whereas increased leukocyte HIF-1α may be responsible for increased inflammation. To elucidate the specific role of leukocyte HIF-1α in COPD, we exposed transgenic mice with conditional deletion or overexpression of HIF-1α in leukocytes to cigarette smoke for 7 mo. Outcomes included pulmonary physiology, aerated lung volumes via microcomputed tomography, lung morphometry and histology, and cardiopulmonary hemodynamics. On aggregate, cigarette smoke increased the aerated lung volume, quasi-static lung compliance, inspiratory capacity of all strains while reducing the total alveolar septal volume. Independent of smoke exposure, mice with leukocyte-specific HIF-1α overexpression had increased quasi-static compliance, inspiratory capacity, and alveolar septal volume compared with mice with leukocyte-specific HIF-1α deletion. However, the overall development of cigarette smoke-induced lung disease did not vary relative to control mice for either of the conditional strains. This suggests that the development of murine cigarette smoke-induced airspace disease occurs independently of leukocyte HIF-1α signaling.

Isolation and Analysis of Macrophage Subsets from the Mouse and Human Lung.

Pulmonary macrophages are heterogeneous. Distinct populations of resident tissue macrophages exist in the lung airspace and tissue compartments during homeostasis. During inflammation, these are joined by monocyte-derived recruited macrophages. Flow cytometry can be used to identify and purify lung macrophage subsets. Here, we describe methods for identifying and isolating macrophages from bronchoalveolar lavage and digested lung tissues from mouse and human. We also describe basic staining for flow cytometry analysis of different macrophage subsets.

Localization of Macrophages in the Human Lung via Design-based Stereology.

Rationale: Interstitial macrophages (IMs) and airspace macrophages (AMs) play critical roles in lung homeostasis and host defense, and are central to the pathogenesis of a number of lung diseases. However, the absolute numbers of macrophages and the precise anatomic locations they occupy in the healthy human lung have not been quantified.Objectives: To determine the precise number and anatomic location of human pulmonary macrophages in nondiseased lungs and to quantify how this is altered in chronic cigarette smokers.Methods: Whole right upper lobes from 12 human donors without pulmonary disease (6 smokers and 6 nonsmokers) were evaluated using design-based stereology. CD206 (cluster of differentiation 206)-positive/CD43+ AMs and CD206+/CD43- IMs were counted in five distinct anatomical locations using the optical disector probe.Measurements and Main Results: An average of 2.1 × 109 IMs and 1.4 × 109 AMs were estimated per right upper lobe. Of the AMs, 95% were contained in diffusing airspaces and 5% in airways. Of the IMs, 78% were located within the alveolar septa, 14% around small vessels, and 7% around the airways. The local density of IMs was greater in the alveolar septa than in the connective tissue surrounding the airways or vessels. The total number and density of IMs was 36% to 56% greater in the lungs of cigarette smokers versus nonsmokers.Conclusions: The precise locations occupied by pulmonary macrophages were defined in nondiseased human lungs from smokers and nonsmokers. IM density was greatest in the alveolar septa. Lungs from chronic smokers had increased IM numbers and overall density, supporting a role for IMs in smoking-related disease.

Polymerizable superoxide dismutase mimetic protects cells encapsulated in poly(ethylene glycol) hydrogels from reactive oxygen species-mediated damage.

A polymerizable superoxide dismutase mimetic (SODm) was incorporated into poly(ethylene glycol) (PEG) hydrogels to protect encapsulated cells from superoxide-mediated damage. Superoxide and other small reactive oxygen species (ROS) can cause oxidative damage to donor tissue encapsulated within size exclusion barrier materials. To enzymatically breakdown ROS within biomaterial cell encapsulation systems, Mn(III) Tetrakis[1-(3-acryloxy-propyl)-4-pyridyl] porphyrin (MnTTPyP-acryl), a polymerizable manganese metalloporphyrin SOD mimetic, was photopolymerized with PEG diacrylate (PEGDA) to create functional gels. In unmodified PEG hydrogels, a significant reduction in metabolic activity was observed when encapsulated Min6 ß-cells were challenged with chemically generated superoxide. Cells encapsulated within MnTPPyP-co-PEG hydrogels, however, demonstrated greatly improved metabolic activity following various superoxide challenges. Further, cells were encapsulated and cultured for 10 days within MnTPPyP-co-PEG hydrogels and challenged with superoxide on days 4, 6, and 8. At the conclusion of this study, cells in blank PEG hydrogels had no observable metabolic activity but when encapsulated in MnTPPyP-functionalized hydrogels, cells retained 60 ± 5% of the metabolic activity compared to untreated controls.

Inducing local T cell apoptosis with anti-Fas-functionalized polymeric coatings fabricated via surface-initiated photopolymerizations.

Cell encapsulation has long been investigated as a means to achieve transplant immunoprotection as it creates a physical barrier between allograft tissue and host immune cells. Encapsulation with passive barrier materials alone, however, is generally insufficient to protect donor tissue from rejection, because small cytotoxic molecules produced by activated T cells can diffuse readily into the capsule and mediate allograft death. As a means to provide bioactive protection for polymeric encapsulation devices, we investigated a functionalized polymeric coating that mimics a natural T cell regulation pathway. T cells are regulated in vivo via Fas, a well-known 'death receptor,' whereby effector cells express Fas ligand and elicit T cell apoptosis upon binding the Fas receptor on a T cell surface. Anti-Fas antibodies are capable of replicating this effect and induce T cell apoptosis in solution. Here, an iniferter-based living radical polymerization was utilized to fabricate surface-anchored polymer chains containing poly(ethylene glycol) with covalently incorporated pendant anti-Fas antibody. Using this reaction mechanism, we demonstrate fabrication conditions that yield surface densities in excess of 1.5 ng/cm(2) of incorporated therapeutic, as detected by ELISA. Additionally, we show that coatings containing anti-Fas antibody induced significant T cell apoptosis, 21+/-2% of cells, after 24h. Finally, the incorporation of a T cell adhesion ligand, intracellular adhesion molecule-1, along with anti-Fas antibody, yielded even higher levels of apoptosis, 34+/-1% of T cells, compared to either signal alone.