BALO: a “mini lung” model to study cell–cell interactions

Abstract

Stem cell‐derived organoid models have emerged as a valuable tool for studying organogenesis, cell‐to‐cell stromal communication and disease. In this issue, Vazquez‐Armendariz et al (2020) report a murine lung stem cell‐based bronchioalveolar organoid system and provide insights into the effect of co‐culturing with immune and mesenchymal cells.

New work reports organoids to analyse stromal‐epithelial crosstalk in the bronchio‐alveolar compartment of the lungs

Direct exposure of the human lung to environment makes it vulnerable to injuries, which can lead to respiratory diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). Remarkably, the in vivo mouse injury models have greatly advanced our understanding of the repair and regeneration of the lung (Hogan et al, 2014; Tata & Rajagopal, 2017). Over the years, multiple studies have identified tissue‐resident stem/progenitor cells across the trachea‐bronchial tree and the alveolar regions of the lung that participate in regeneration after injury. In the airways, basal stem cells and secretory cells serve as the main source of cells that can replenish lost cells. In the alveoli, alveolar type‐2 cells (AT2) are the primary source of cells that can repair damaged epithelium. In addition, a rare population of cells that co‐express SFTPC and SCGB1A1, markers of AT2 and secretory cells, respectively, and reside at the bronchioalveolar duct junction (BADJ) has the ability to contribute to both airway and alveolar epithelium. Specifically, earlier work using purified BASCs in ex vivo cultures (Giangreco et al, 2002; Kim et al, 2005) and more recent work using intersectional transgenics (Liu et al, 2019; Salwig et al, 2019) have shown that BASCs can self‐renew and contribute to the club and ciliated cells, alveolar epithelial cells during regeneration after injury. Despite these advances, we currently do not have the complete understanding of complex cell–cell interactions and how they influence stem cell fate in normal, regenerating and disease tissue contexts. For example, how do different stromal cells influence BASC proliferation and cell fate decisions?

To understand such complex cell–cell interactions, organoids have emerged as valuable models as they recapitulate features of complex tissues. In addition, these three‐dimensional, self‐organized tissue‐like structures allow for studying morphogenesis and disease in ex vivo cultures. Isolated stem/progenitor cells from both airways and alveoli have the ability to generate spheroids/organoids (Barkauskas et al, 2017; Lee et al, 2017; Katsura et al, 2019). To date, these lung organoids are limited in their complexity that they do not recapitulate the complex lung microenvironment and do not include immune and endothelial compartments. In the current issue, Vazquez‐Armendariz et al (2020) report development of a complex mouse bronchioalveolar lung organoid (BALO) system by co‐culturing bronchioalveolar stem cells (BASCs) with defined subsets of lung‐resident mesenchymal cells (rMCs). Subsequently, this study also incorporated tissue‐resident macrophages and explored their effect on epithelial differentiation and maturation in BALO cultures (Fig 1).

Figure 1. Schematic representation of BALO culture model.

Bronchioalveolar stem cells (BASCs) were cultured with macrophages or different mesenchymal cells in Matrigel‐based environments. BASCs generated different outcome depending on the co‐culture model.

The authors first sought to identify an epithelial cell population that is competitive to generate both airway and alveolar cells. Using EpCAM^highCD24^lowSca‐1⁺, the authors purified bronchioalveolar stem cells that have been previously shown to generate both airway and alveolar cells when co‐cultured with foetal endothelial cells (Lee et al, 2017). Further, the authors tested the potential of EpCAM^highCD24^lowSca‐1⁺cells in the presence of Sca‐1⁺PDGFRα^low/high cells, which account for most of the lung‐resident mesenchymal cells. The authors observed highly branched 3D organoid structures within 21 days, which mimics the cellular composition of bronchiolar‐like and alveolar‐like lung structures. In addition, Vazquez‐Armendariz and colleagues also isolated SCGB1A1⁺SFTPC⁺ bronchioalveolar stem cells (BASCs) from two newly generated mouse models that express fluorescent reporters under the control of SCGB1A1 and SFTPC promoters (Salwig et al, 2019). Using this model, the authors showed that of all the organoids derived from EpCAM^highCD24^lowSca‐1⁺ cells were predominantly originated from BASCs.

In addition to the structural framework, lung mesenchyme constitutes an important stem cell niche and regulates stem cell fate during development and disease (Zepp et al, 2017; Basil et al, 2020). To test whether distinct mesenchymal cells influence the growth of BALO, the authors utilized Pdgfra ^GFP reporter mouse line to isolate two populations of resident mesenchymal cells (rMCs) based on PDGFRα and Sca‐1 expression (Sca‐1^highPDGFRα^low and Sca‐1^intPDGFRα^high). Intriguingly, while Sca‐1^highPDGFRα^low rMCs supported organoid growth but failed to induce cell differentiation and branching morphogenesis, in contrast, Sca‐1^intPDGFRα^high rMCs alone were not sufficient to support organoid growth but were able to induce epithelial differentiation and branching morphogenesis. Further, immunofluorescence analysis suggested that LipidTOX⁺ PDGFRα^low lipofibroblasts are distributed around developing organoids whereas αSMA⁺ PDGFRα^high myofibroblasts seem to localize at branching sites. While the current study uncovered distinct functions for the two rMC populations, it is unclear whether they constitute anatomically and functionally distinct populations in vivo? Future studies will need to directly compare the relationship between the two rMCs with that of previously identified distinct peri‐bronchiolar and alveolar mesenchymal cells (Lee et al, 2017; Zepp et al, 2017). In addition, it is equally important to map the spatial localization of the two rMCs and how they interact with epithelium in a spatiotemporal manner to regulate the BASC turnover during homeostasis, injury, repair and regeneration.

Macrophages form another essential component of the alveolar stem cell niche and exert different roles during branching, alveolarization and repair after injury (Lechner et al, 2017). To study the epithelial–immune cell interactions, the authors established a microinjection protocol to successfully “engraft” tissue‐resident macrophages (TR‐Macs) derived from bronchioalveolar lavage (BAL) fluid of adult mice into 14 days BALO compartments. Electron microscopy data demonstrated a direct contact between TR‐Mac and epithelial cells via filopodia‐like protrusions extending from TR‐Mac to epithelial cells. Additionally, the authors found that TR‐Macs uptake and digest surfactants produced by the epithelial cells. Interestingly, TR‐Macs established a direct communication with epithelial cells as revealed by Connexin‐43 expression in TR‐Macs in co‐culture model. Cx43 expression was not observed in TR‐Macs without BALO, indicating that both epithelial and TR‐Macs established a bidirectional communication in BALO cultures. Furthermore, single‐cell RNA sequencing (scRNA‐seq) data demonstrated that the addition of TR‐Macs drives BALO differentiation by suppressing the cell proliferation markers Fos, Fosb, Areg and Klf4 and accelerate the maturation of BALO by inducing the cell differentiation markers such as Neat1, Cyp2f2 and Ces1d. Additionally, electron microscopy (EM) assisted ultra‐structural analysis demonstrated that BALOs form airway‐like branched tissues. BALO consists of differentiated secretory cells filled with secretory granules, ciliated cells with mature cilia and basal bodies aligned underneath the apical cell surface, and AT1 and AT2 cells. Notably, the authors also point to cells that appear to reside basally and morphologically similar to basal cells. To further validate these findings, the authors used scRNA‐seq and found both alveolar and airway epithelial cells, including basal cells in BALO‐derived cultures. The finding that BASC‐derived organoid cultures can generate basal cells is in stark contrast to recent lineage tracing studies in vivo (Liu et al, 2019). Whether this observation is limited to the in vitro plasticity remains to be studied. Nevertheless, the plasticity and the multipotency of BASCs uncovered here will be valuable for future cell‐based regenerative therapies.

Finally, the authors applied the epithelial‐derived BALO to study the influenza virus infection using the SC35M‐GFP reporter virus. Microinjection of the reporter virus suspension into BALO allowed them to map the spread of viral infection. Moreover, the authors showed injection of TR‐Macs in BALO increases the release of the pro‐inflammatory cytokines TNFα, IL‐1β and IL‐6 compared to BALO without TR‐Macs.

Together, BALO is a complex organoid model that provides an opportunity to study the epithelial–mesenchymal–immune cell interactions ex vivo. Additionally, BALO opens a new model system to interrogate the cellular responses after injury or infection ex vivo. Currently, the BALO model does not incorporate endothelial and other immune cells, which are an integral part of the respiratory system. Therefore, further development of BALO model is essential to realize an ideal ex vivo platform to study morphogenesis and disease. In addition, BALO model may not be directly applicable to modelling human lung branching morphogenesis and disease ex vivo as BASCs equivalent population has not been identified in adult human lungs. Nevertheless, BALO offers a model to study these processes in murine models, and perhaps, recent emergence of high‐throughput single‐cell profiling studies coupled with BALO‐like models may find BASCs equivalent multipotent progenitor population in the human lung.

The EMBO Journal (2020) 39: e106697