Chronic and acute lung diseases are amongst the leading causes of morbidity worldwide (Soriano et al., 2020). Due to their prevalence and severity, which is further exacerbated with aging, these diseases require further understanding from a cellular and molecular perspective. The lung encapsulates a variety of cell niches including the trachea, bronchioles, and alveolar space, which are supported by the smooth muscles, fibroblasts, and endothelium. When analyzing the varying lung diseases such as bronchopulmonary dysplasia, cystic fibrosis, chronic obstructive pulmonary disease, pulmonary fibrosis, and SARS-COV-2, one common thread of lung disease is a depletion or dysfunction of lung alveolar epithelial cells, bronchiolar or airway epithelial cells, or both (Figure 1). Therefore, better understanding how lung cell niches protect and repair the epithelial cells may allow us to combat these chronic and acute lung conditions. To a large extent the specialized niches control stem cell self-renewal and differentiation in the lung (Lee et al., 2014). It has been previously shown that lung repair processes are mediated via progenitor cells in the lung. Therefore, these progenitor cells serve an important function in the lung microenvironment. There is an abundance of progenitor cells in the lung, such as basal cells in the trachea, Alveolar type II cells (AT2 cells) in the alveolar space, Club cells in the bronchioles, and many others. In order to better understand lung disease emergence and progression, as well as its potential repair process, it is important to further understand these important progenitor cells.
Previous work in our lab has demonstrated the ability of organoids to model the progenitor cell properties of alveolar and bronchiolar cell types (Lee et al., 2014; Kim et al., 2005). The three-dimensional (3D) organoid co-culture system we developed involves the co-culturing of epithelial EPCAM+ cells (SCA1- or SCA1+) with supporting lung mesenchymal cells in Matrigel. These 3D culture systems are able to mimic the lung niche and advance the understanding of lung biology. SCA1+ lung epithelial cells give rise to both alveolar and bronchiolar organoids, modeling the progenitor cell capacity of multipotent bronchioalveolar stem cells (BASCs), whereas SCA1- lung epithelial cells give rise to only alveolar organoids, modeling the capacity of AT2 cells. In the context of oncogenic Kras expression, AT2 cell organoids mimic tumor progression in vivo tumor progression and recapitulate early-stage lung adenocarcinoma in patients (Figure 3)(Dost et al., 2020). This resource could aid in identifying transcriptional and proteomic differences that distinguish normal epithelium from lung cancer. Thus, organoids serve as a useful tool to model numerous lung diseases and understand their mechanisms and progression.
Aging is a predominant risk factor for chronic lung diseases and lung cancer. Despite its significant impact, the impact of aging on lung progenitor cell functions remains largely understudied from a cellular perspective. Previous work has demonstrated lung progenitor population changes, compromised repair, and epigenetic instability as a result of aging in mice (Schneider et al., 2021). Specifically, in our laboratory, we have seen a change in the frequency of lung alveolar progenitor cells with aging. The changes largely show a shift toward significantly fewer AT2 cells and significantly more bronchiolar progenitors including BASCs (Rowbotham et al., bioRxiv, 2021). Additionally, changes in organoid-forming efficiency and typology have been demonstrated with age. Organoids grown from SCA1- epithelial cells from old mice yielded a lower alveolar colony-forming efficiency than organoids grown from cells taken from young mice. A similar trend was observed when analyzing organoids grown from SCA1+ epithelial cells with old mice yielding a lower alveolar colony-forming efficiency than young mice. However, organoids grown from SCA1+ epithelial cells from old mice yielded a higher bronchiolar organoid-forming efficiency.
Besides the effects aging has on lung progenitor cells, we found aging causes epigenetic alterations, such as a decrease in Lysine 9 methylation. This was indicated by H3K9me2 fluorescence data showing a lower fluorescence value for old mice as compared to young mice. Lysine 9 methylation depletion modeling through use of an inhibitor of G9a, the methyltransferase that bi- and tri-methylates lysine 9, reduced alveolar progenitor activity in organoid cultures. Aging has also been shown to increase lung damage post alveolar injury via bleomycin (Hecker et al., 2014). We have recapitulated this phenotype with depletion of Lysine 9 methylation in young mice by G9a inhibitor administration. Bleomycin-injured mice suffered greater persistent lung damage when G9a inhibitors were administered. Interestingly, mice depleted of Lysine 9 methylation showed an expansion of bronchiolar progenitors and a decrease in alveolar progenitors, suggesting that, at the expense of alveolar progenitors, bronchiolar progenitor cell activity is enhanced. All of these differences demonstrate how one key aspect of aging in the lung may be the abrogation of epigenetic regulation mediated by G9a, which changes the dynamics of how different progenitor cell types are used in repair and regeneration (Rowbotham et al., bioRxiv, 2021).
Recent studies in lung biology have identified lung progenitor cells that may one day be used to treat varying pulmonary diseases (Barkauskas et al., 2013; Hong et al., 2001; Kathiriya et al., 2020; Kim et al., 2005; Rawlins et al., 2009; Louie et al., 2022). Current treatment modalities for patients suffering from pulmonary diseases largely rely on whole or partial lung transplantation from donor lungs. Due to the shortage of donor lungs, an alternative to whole and partial lung transplantations would be useful in combating these varying pulmonary diseases. A potential alternative may be to utilize lung progenitor cells as a treatment modality. For example, alveolar type I and AT2 cells may be useful in treating idiopathic pulmonary fibrosis, as these are the lung cell types impacted throughout the course of this disease. Our laboratory has tested the potential of lung organoid cells to be transplanted into the lung while retaining progenitor status, a critical aspect for use in future treatment modalities (Louie et al., 2022). We conducted a multi-faceted study, analyzing the engraftment of these cells, their gene expression program via single-cell RNA sequencing, and their proliferative potential after re-injury in recipient mice. We transplanted organoids derived from Sca1- cells and organoids derived from Sca1+ cells into bleomycin injured mice. We then analyzed mice at varying time points and investigated the transplanted cells via flow analysis, immunohistochemical staining, and RNA sequencing. Transplanted AT2 cells expressed the AT2 marker surfactant protein C (SPC), suggesting engraftment of the organoid cells (Figure 5). Single cell RNA sequencing revealed that transplanted and native cells were transcriptionally similar but distinct from the organoid cluster. Furthermore, flow cytometry analysis and immunostaining showed a similar proliferation potential for transplanted cells and native AT2 cells in the lung (Louie et al., 2022). In conclusion, we were able to show that alveolar cells maintained in organoid cultures retain progenitor cell activity after transplantation, an important concept for their potential future use in therapy.
Besides the relevant uses of the organoid system as a tool for investigating lung cancer and disease using mouse models, there have been many new advances that shed new light on the identity of human lung progenitor cells and how to maintain the diversity of lung cell types in organoid cultures. For example, recent work has identified progenitor cell populations in anatomical structures that only exist in the human lung (Basil et al., 2022, Murthy et al., 2022). Such discoveries and many more are required to understand the complexity of human lung biology and how to intervene when lung homeostasis goes awry in a clinical setting. Organoids may serve as a useful and feasible treatment modality for lung diseases in the future, depending on the injury and cell populations impacted. Alveolar organoids provide treatment for surfactant protein deficiency, Hermansku-Pudlak Syndrome, pulmonary fibrosis, and bronchopulmonary dysplasia. Airway organoids may help in the treatment of chronic obstructive pulmonary disease, cystic fibrosis, and bronchiolitis. Future treatment options made possible through organoid modeling or organoid transplantations have the potential to treat a variety of chronic and acute diseases, impacting many patients worldwide.
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