Organoids are essentially an in vitro (lab-grown) three-dimensional tissue culture system model that meticulously replicates the structure and physiology of human organs. A few of the present applications of organoids are in the basic biological research area, molecular medicine and pharmaceutical drug testing. Organoids are crucial in connecting the gap between animal models and human clinical trials during the drug discovery process, which significantly lowers the time duration and cost associated with each stage of testing. Likewise, they can be used to understand cell-to-cell interactions, a crucial aspect of tissue biology and regeneration, and to model disease pathogenesis at various stages of the disease. Lung organoids can be utilized to explore numerous pathophysiological activities of a lung since they share similarities with its function. Researchers have been trying to recreate the complex nature of the lung by developing various \"Lung organoids\" models.This article is a systematic review of various developments of lung organoids and their potential progenitors. It also covers the in-depth applications of lung organoids for the advancement of translational research. The review discusses the methodologies to establish different types of lung organoids for studying the regenerative capability of the respiratory system and comprehending various respiratory diseases.Respiratory diseases are among the most common worldwide, and the growing burden must be addressed instantaneously. Lung organoids along with diverse bio-engineering tools and technologies will serve as a novel model for studying the pathophysiology of various respiratory diseases and for drug screening purposes.

Durable reconstitution of the distal lung epithelium with pluripotent stem cell (PSC) derivatives, if realized, would represent a promising therapy for diseases that result from alveolar damage. Here, we differentiate murine PSCs into self-renewing lung epithelial progenitors able to engraft into the injured distal lung epithelium of immunocompetent, syngeneic mouse recipients. After transplantation, these progenitors mature in the distal lung, assuming the molecular phenotypes of alveolar type 2 (AT2) and type 1 (AT1) cells. After months in vivo, donor-derived cells retain their mature phenotypes, as characterized by single-cell RNA sequencing (scRNA-seq), histologic profiling, and functional assessment that demonstrates continued capacity of the engrafted cells to proliferate and differentiate. These results indicate durable reconstitution of the distal lung\'s facultative progenitor and differentiated epithelial cell compartments with PSC-derived cells, thus establishing a novel model for pulmonary cell therapy that can be utilized to better understand the mechanisms and utility of engraftment.

Sex differences exist for many lung pathologies, including COVID-19 and pulmonary fibrosis, but the mechanistic basis for this remains unclear. Alveolar type 2 cells (AT2s), which play a key role in alveolar lung regeneration, express the X-linked Ace2 gene that has roles in lung repair and SARS-CoV-2 pathogenesis, suggesting that X chromosome inactivation (XCI) in AT2s might impact sex-biased lung pathology. Here we investigate XCI maintenance and sex-specific gene expression profiles using male and female AT2s. Remarkably, the inactive X chromosome (Xi) lacks robust canonical Xist RNA \"clouds\" and less enrichment of heterochromatic modifications in human and mouse AT2s. We demonstrate that about 68% of expressed X-linked genes in mouse AT2s, including Ace2, escape XCI. There are genome-wide expression differences between male and female AT2s, likely influencing both lung physiology and pathophysiologic responses. These studies support a renewed focus on AT2s as a potential contributor to sex-biased differences in lung disease.

The respiratory epithelium consists of multiple, functionally distinct cell types and is maintained by regionally specific progenitor populations that repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine lung cells, but isolation methods are hampered by a lack of surface markers distinguishing epithelial progenitors along the respiratory epithelium. Here, we developed a 3D printed lobe divider (3DLD) to aid in simultaneous isolation of proximal versus distal lung epithelial progenitors from individual mice that give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, commonly used manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal cells with distal cells, which altered the transcriptional landscape and size distribution of distal organoids. The 3DLD aids in reproducible isolation of distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays.

Tobacco smoking has been established to contribute to the pathogenesis of various respiratory diseases including chronic obstructive pulmonary disease (COPD), lung cancer, and asthma. However, major hurdles in mechanistic studies on the role of smoking in human lungs remain in part due to the lack of ex vivo experimental models and ambiguous data from animal models that can best recapitulate the architecture and pathophysiology of the human lung. Recent development of the lung organoid culture system has opened new avenues for respiratory disease research as organoids are proving to be a sophisticated ex vivo model that functionally and structurally mimics the human lungs better than other traditionally used models. This review will discuss how recent advances in lung organoid systems may help us better determine the injurious and immunological effect of smoking on human lungs and will provide some suggestions for future research directions.

BACKGROUND: A healthy alveolar epithelium is critical to the gas exchange function of the lungs. As the major cell type of alveolar epithelium, alveolar type 2 (AT2) cells play a critical role in maintaining pulmonary homeostasis by serving as alveolar progenitors during lung injury, inflammation, and repair. Dysregulation of AT2 cells may lead to the development of acute and chronic lung diseases and cancer. The lack of clinically relevant AT2 cell models hampers our ability to understand pulmonary diseases. Here, we sought to establish reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2) and investigate their potential in forming alveolar organoids to model pulmonary diseases.
METHODS: Primary mouse pulmonary alveolar cells (mPACs) were isolated and immortalized with a retroviral expression of SV40 Large T antigen (LTA). Cell proliferation and survival was assessed by crystal violet staining and WST-1 assays. Marker gene expression was assessed by qPCR, Western blotting, and/or immunostaining. Alveolar organoids were generated by using matrigel. Ad-TGF-β1 was used to transiently express TGF-β1. Stable silencing β-catenin or overexpression of mutant KRAS and TP53 was accomplished by using retroviral vectors. Subcutaneous cell implantations were carried out in athymic nude mice. The retrieved tissue masses were subjected to H & E histologic evaluation.
RESULTS: We immortalized primary mPACs with SV40 LTA to yield the imPACs that were non-tumorigenic and maintained long-term proliferative activity that was reversible by FLP-mediated removal of SV40 LTA. The EpCAM+ AT2-enriched subpopulation (i.e., imPAC2) was sorted out from the imPACs, and was shown to express AT2 markers and form alveolar organoids. Functionally, silencing β-catenin decreased the expression of AT2 markers in imPAC2 cells, while TGF-β1 induced fibrosis-like response by regulating the expression of epithelial-mesenchymal transition markers in the imPAC2 cells. Lastly, concurrent expression of oncogenic KRAS and mutant TP53 rendered the imPAC2 cells a tumor-like phenotype and activated lung cancer-associated pathways. Collectively, our results suggest that the imPAC2 cells may faithfully represent AT2 populations that can be further explored to model pulmonary diseases.

The 2022 Annual Review Issue of The Journal of Pathology, Recent Advances in Pathology, contains 15 invited reviews on research areas of growing importance in pathology. This year, the articles include those that focus on digital pathology, employing modern imaging techniques and software to enable improved diagnostic and research applications to study human diseases. This subject area includes the ability to identify specific genetic alterations through the morphological changes they induce, as well as integrating digital and computational pathology with \'omics technologies. Other reviews in this issue include an updated evaluation of mutational patterns (mutation signatures) in cancer, the applications of lineage tracing in human tissues, and single cell sequencing technologies to uncover tumour evolution and tumour heterogeneity. The tissue microenvironment is covered in reviews specifically dealing with proteolytic control of epidermal differentiation, cancer-associated fibroblasts, field cancerisation, and host factors that determine tumour immunity. All of the reviews contained in this issue are the work of invited experts selected to discuss the considerable recent progress in their respective fields and are freely available online (https://onlinelibrary.wiley.com/journal/10969896). © 2022 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The human lung is a relatively quiescent organ in the normal healthy state but contains stem/progenitor cells that contribute to normal tissue maintenance and either repair or remodeling in response to injury and disease. Maintenance or repair lead to proper restoration of functional lung tissue and maintenance of physiological functions, with remodeling resulting in altered structure and function that is typically associated with disease. Knowledge of cell types contributing to lung tissue maintenance and repair/remodeling have largely relied on mouse models of injury-repair and lineage tracing of local progenitors. Therefore, many of the functional alterations underlying remodeling in human lung disease have remained poorly defined. However, the advent of advanced genomics approaches to define the molecular phenotype of lung cells at single-cell resolution has paved the way for rapid advances in our understanding of cell types present within the normal human lung and changes that accompany disease. Here we summarize recent advances in our understanding of disease-related changes in the molecular phenotype of human lung epithelium that have emerged from single-cell transcriptomic studies. We focus attention on emerging concepts of epithelial transitional states that characterize the pathological remodeling that accompanies chronic lung diseases, including idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, and asthma. Concepts arising from these studies are actively evolving and require corroborative studies to improve our understanding of disease mechanisms. Whenever possible, we highlight opportunities for providing a unified nomenclature in this rapidly advancing field of research. © 2022 The Pathological Society of Great Britain and Ireland.

Over the last decades, several studies demonstrated the possibility of lung regeneration through transplantation of various lung progenitor populations. Recently, we showed in mice that fetal or adult lung progenitors could potentially provide donor cells for transplantation, provided that the lung stem cell niche in the recipient is vacated of endogenous lung progenitors by adequate conditioning. Accordingly, marked lung regeneration could be attained following i.v. infusion of a single cell suspension of lung cells into recipient mice conditioned with naphthalene (NA) and 6Gy total body irradiation (TBI). As clinical translation of this approach requires the use of allogenic donors, we more recently developed a novel transplantation modality based on co-infusion of hematopoietic and lung progenitors from the same donor. Thus, by virtue of hematopoietic chimerism, which leads to immune tolerance toward donor antigens, the lung progenitors can be successfully engrafted without any need for post-transplant immune suppression. In the present study, we demonstrate that it is possible to replace NA in the conditioning regimen with Cyclophosphamide (CY), approved for the treatment of many diseases and that a lower dose of 2 GY TBI can successfully enable engraftment of donor-derived hematopoietic and lung progenitors when CY is administered in 2 doses after the stem cell infusion. Taken together, our results suggest a feasible and relatively safe protocol that could potentially be translated to clinical transplantation of lung progenitors across major MHC barriers in patients with terminal lung diseases.

Lung transplantation is the only treatment available for end-stage lung diseases; however, donor shortage is a global issue. The use of human pluripotent stem cells (hPSCs) for organ regeneration is a promising approach. Nevertheless, methods for the expansion of isolated hPSC-derived lung progenitors (hLPs) for transplantation purposes have not yet been reported. Herein, we established an expansion system of hLPs based on their three-dimensional culture in core-shell hydrogel microfibers, that ensures the maintenance of their bipotency for differentiation into alveolar and airway epithelial cells including alveolar type II (AT2) cells. Further, we developed an efficient in vivo transplantation method using an endoscope-assisted transtracheal administration system; the successful engraftment and in vivo differentiation of hLPs into alveolar epithelial cells (incorporated into the alveoli) was observed. Importantly, expanded hLPs in the context of microfibers were successfully transplanted into the murine lungs, opening avenues for cell-based therapies of lung diseases. Therefore, our novel method has potential regenerative medicine applications; additionally, the high-quality hLPs and AT2 cells generated via the microfiber-based technology are valuable for drug discovery purposes.