COVID-19: Researchers create human airway stem cells from patients’ cells

For the first time, researchers have successfully created airway basal stem cells in vitro from induced pluripotent stem cells by reprogramming blood cells taken from patients.

Given that airway basal cells are defined as stem cells of the airways because they can regenerate the airway epithelium in response to injury, this study may help accelerate research on diseases impacting the airway, including COVID-19, influenza, asthma and cystic fibrosis.

Led by researchers at the Center for Regenerative Medicine at Boston Medical Center and Boston University (CReM), in collaboration with The University of Texas Health Science Center at Houston (UTHealth), these findings represent a critical first step towards airway regeneration, which will advance the field of regenerative medicine as it relates to airway and lung diseases.

Published in Cell Stem Cell, the novel study outlines how to efficiently generate and purify large quantities of airway basal stem cells using patient samples.

This allows for the development of individual, disease-specific airway basal stem cells in a lab that can be used to develop disease models, which may ultimately lead to drug development and a platform in which targeted drug approaches can be tested.

The study’s findings and cells will be shared freely given the CReM’s “Open Source Biology” philosophy, or sharing of information and findings that will help advance science across the globe.

“Simply put, we have developed a way to reproduce patient-specific airway basal cells in the lab, with the ultimate goal of being able to regenerate the airway for patients with airway diseases,” said Finn Hawkins, MB, BCh, a pulmonologist and physician-scientist at Boston Medical Center, principal investigator in the CReM and the Pulmonary Center and the study’s first author.

“These results could lead to a better understanding, and therefore treatments for, a variety of airway diseases,” said Shingo Suzuki, Ph.D., co-first author and post-doctoral researcher at UTHealth. For example, cystic fibrosis is caused by a genetic mutation that is present in all of the airway cells.

“If we could make pluripotent stem cells using a sample from a patient who has cystic fibrosis, correct the mutation and replace the defective airway cells with corrected airway basal cells that are otherwise genetically identical, we might eventually be able to cure the disease, and other diseases in the future using this same technology,” added Hawkins.

Induced pluripotent stem cells are the master stem cells that can produce any cell or tissue in the human body. They are created by reprogramming a human sample, such as a drop of blood, into a population of cells that are similar to embryonic stem cells, including the ability to form different cell types within organs.

For this study, the researchers established methods to generate an airway stem cell population, basal cells in the laboratory. These are an important cell type in human airways that maintain the lining of cells in the airway, including the cells the make mucus and those that propel the mucus upwards and out of the lungs.

The researchers first engineered induced pluripotent stem cells with a genetic sequence encoding a fluorescent protein that would allow them to visualize, track and purify basal cells if present.

Then, the researchers turned to studies of the embryo and prior work in this field to determine how basal form as the lungs develop. By manipulating induced pluripotent stem cells with a series of steps aimed to simulate what happens during lung development the researchers successfully generated cells that were highly similar to human airway basal cells in terms of their appearance, the genes they expressed and most importantly, their ability to both proliferate and form the other cell types of the airway.

The cells, termed ibasal cells, were able to regenerate an airway in vivo using a rodent trachea model.

The resulting ibasal cells, made from patients with a variety of lung diseases, were also able to model the airway diseases affecting those patients, including the mucus metaplasia that is characteristic of asthmatic airways, the chloride channel dysfunction that causes cystic fibrosis, and the defects in beating of the cilia that causes the disease primary ciliary dyskinesia.

This approach will enable future opportunities to study these genetic changes, and how to reverse them in order to cure the disease in humans. From a practical perspective, ibasal cells grow well in special culture conditions in a lab, allowing them to be made in large numbers, and patient specific basal cells can be grown, frozen for future work, and shared with the broader research community.

“We demonstrated the potential of these ibasal cells to model both human development and disease, providing evidence of their capacity to regenerate airway epithelium,” said Hawkins, who is also an assistant professor of medicine at Boston University School of Medicine.

“We expect this will be a significant breakthrough and will contribute to new insights and treatment options for airway diseases, as our results have overcome several important hurdles currently limiting progress in the field.”

---

The respiratory system is organized into multiple integrated compartments comprising multiple tissues that perform gas exchange between the blood and the external environment. The various anatomical regions of the respiratory tract are populated by numerous types of unique epithelial, vascular, mesenchymal, and immune cells critical for the functioning of each particular compartment.

Historically, the development of the respiratory system has been thought to involve several discrete morphogenetic steps including lineage specification, branching morphogenesis, sacculation, and alveologenesis (Morrisey and Hogan, 2010). While these steps were previously conceived of in terms of distinct temporal stages of development, more recent evidence has suggested that there is overlap between these stages and particular events such as cell specification and commitment, which are now thought to occur very early and coincident with the basic patterning of the respiratory airway tree (Frank et al., 2019).

The branched network of airways and gas exchange surfaces co-develops with the cardiovascular system to bring both organ systems into intimate proximity for full functionality. More details on these important developmental events can be found in several recent reviews (Herriges and Morrisey, 2014, Hines and Sun, 2014, Morrisey and Hogan, 2010, Nikolić et al., 2018, Whitsett et al., 2019, Zepp and Morrisey, 2019). The culmination of these events is the generation of an extensive surface area for efficient gas exchange that in the human lung comprises approximately 70 m2.

This review will focus on how the mature respiratory system maintains its normal homeostatic structure and function and how it responds to injury and regenerates itself. We will explore the cellular constituents of the two major compartments in the lungs—the gas exchange alveoli and the conducting airways including the trachea—and describe established and emerging techniques to explore human lung regeneration.

Compartment-Specific Regeneration in the Respiratory System
Alveolar Regeneration

The lung alveolus is composed of multiple epithelial, endothelial, and mesenchymal cell types (Figure 1 ). In addition to these resident cell types, the alveolus also is inhabited by several immune cell lineages, including alveolar macrophages, interstitial macrophages, and dendritic cells and several recent datasets have shown this diversity of cells at single-cell resolution in both animals and humans (Guo et al., 2019, Travaglini et al., 2019, Vieira Braga et al., 2019).

Emerging data suggest there is some degree of inter-cellular communication between the lineages in this niche, but our understanding of the crosstalk among alveolar cell lineages during homeostasis or regeneration remains poor. The alveolar compartment remains largely quiescent in the uninjured lung, and most cells within this niche exhibit a relatively slow turnover.

After lung injury, multiple alveolar cell types are able to proliferate, and when repair is effective both alveolar structure and function are restored. This ability to react to injury involves both activation of self-renewal as well as differentiation into more mature cell lineages.

The self-renewal and differentiation of various lung epithelial cells are modulated by a growing list of cell types that includes neighboring epithelial cells, mesenchymal cells, airway smooth muscle, neurons and neuroendocrine cells, endothelium, and various leukocyte populations (Barkauskas et al., 2013, Cao et al., 2017, Lechner et al., 2017, Lee et al., 2017, Rafii et al., 2015, Zepp et al., 2017).

These studies have highlighted recurrent themes regarding the signals that can drive alveolar epithelial regeneration, including Wnt signaling.

Alveolar Epithelial Response to Injury. There are two primary lineages in the alveolar epithelium: the alveolar epithelial type 1 (AT1) and type 2 (AT2) cells (Figure 1). AT1 cells cover 95% of the alveolar surface area and are closely juxtaposed with the capillary plexus. AT2s are responsible for generating pulmonary surfactant, which is essential for reducing the surface tension of the alveolar surface area to prevent the lungs from collapsing upon every breath. AT1 and AT2 cells are specified very early in lung development and AT2 cells do not appreciably generate AT1 cells during early postnatal lung growth (Frank et al., 2016, Frank et al., 2019). However, in adult mice, AT2 cells can act as both a self-renewing stem cell like population and regenerating AT1 cells after injury (Barkauskas et al., 2013). A sublineage within the AT2 cell population that expresses the transcriptional target of Wnt signaling, Axin2, has been shown to play a dominant role in repairing the lung alveolus after acute injury (Nabhan et al., 2018, Zacharias et al., 2018). These cells, which have been called alveolar epithelial progenitors or AEPs, preferentially re-enter the cell cycle after injury, self-renew, and regenerate mature AT1 and AT2 cells. AEPs appear primed to enter the cell cycle and they respond robustly to Wnt and Fgf7 signaling (Zacharias et al., 2018). In alveolar development, Wnt-responsive AT2 cells self-renew in the presence of Wnt signaling and differentiate in its absence (Frank et al., 2016), but it is not yet known whether these same signals drive renewal versus differentiation during regeneration. A recent report also shows that AEPs promote metastasis in models of lung cancer (Laughney et al., 2020). AEPs have been identified in the human lung and are responsible for generating the majority of AT2 cell growth in human alveolar organoids (Zacharias et al., 2018). In contrast to AT2s and AEPs, the AT1 cell contribution to alveolar epithelial regeneration is thought to be very limited (Jain et al., 2015). Following the surgical removal of lung tissue in adult mice and other model organisms, new alveoli are formed to compensate for lost alveolar surface area (Buhain and Brody, 1973). This compensatory lung growth after partial pneumonectomy is commonly used as a “sterile” model of lung regeneration. Not surprisingly, this regenerative response involves the coordinated actions of nearly every cell type in the lung including epithelial cells, endothelial cells, mesenchymal cells, and leukocytes (Chen et al., 2012, Jain et al., 2015, Lechner et al., 2017, Rafii et al., 2015). In this model, a small number of cells expressing Hopx+, a marker for the AT1 lineage, were found to both proliferate and, in rare instances, give rise to Sftpc+ AT2 cells (Jain et al., 2015). A recent study revealed heterogeneity within the AT1 cell population where expression of Igfbp2 marked the most mature AT1 cell subtype that lacks differentiation capacity following pneumonectomy (Wang et al., 2018). Although cells expressing AT1 markers proliferate during compensatory lung growth after partial pneumonectomy, whether bona fide AT1 cells are able to contribute to repair after acute lung injury is largely unknown.

Alveolar Endothelial Response to Injury. Effective gas exchange is dependent upon AT1 cell and pulmonary capillary endothelial cell (PCEC) proximity, and successful alveolar regeneration requires re-establishment of this spatial relationship. Following lung injury in rodents, there is rapid proliferation in the microvasculature, with expansion of resident microvascular endothelial progenitor cells (Alvarez et al., 2008). These cells are marked by Cd34 and Cd309, and in organoid culture demonstrate significant vasculogenic capacity. While both the mechanisms of PCEC regeneration and cellular identities within this compartment are incompletely understood, endothelial cells expressing Sox17 have recently been shown to play a role in endothelial regeneration after endotoxic-induced vascular injury (Liu et al., 2019a). Additionally, PCECs enhance alveologenesis following injury, with PCEC-derived Vegfr2 and Fgfr1 mediating epithelial proliferation (Ding et al., 2011). This signaling is thought to be through MMP14, and possibly co-mediated through platelet activation of endothelial SDF-1 receptors (Rafii et al., 2015). Significant additional heterogeneity is thought to exist within the distal lung endothelium, with differential vasculogenic capacities and crosstalk with the epithelium (Stevens et al., 2008), and this has recently been revealed in single-cell transcriptomic studies of the homeostatic lung and its response to acute injury (Niethamer et al., 2020). This study demonstrated the presence of multiple microvascular endothelial subsets including one which expresses high levels of Car4 and Cd34. This study also showed that a population of proliferating endothelial cells emerges soon after influenza injury and single-cell informatic trajectory analysis suggests that these cells arise from multiple endothelial subsets. Future work is needed to define the functional role for these endothelial subsets in both normal alveolar homeostasis and the response to injury.

Alveolar Mesenchymal Response to Injury. The lung alveolus is also home to a complex mixture of mesenchymal cell types, many of which are in close physical association with both alveolar epithelial and endothelial cells and play an active role in alveolar epithelial regeneration. Pioneering electron microscopy studies demonstrated direct and extensive contacts between lung fibroblasts and AT2 cells (Sirianni et al., 2003, Walker et al., 1995). Mesenchymal cells expressing the platelet-derived growth factor alpha (Pdgfra) are often found in close association with AT2 cells (Barkauskas et al., 2013, Green et al., 2016, Zepp et al., 2017). The first functional evidence of trophic interactions between these populations was the observation that Pdgfra+ fibroblasts support the growth and differentiation of AT2s in an alveolar organoid co-culture assay (Barkauskas et al., 2013). Alveolar organoids have been recently used to provide functional evidence that multiple signaling pathways originate in Pdgfra+ cells to influence AT2 cell self-renewal and differentiation into AT1 cells including Fgf7, Bmp, and Il6 (Chung et al., 2018, Green et al., 2016, Zepp et al., 2017). As molecular techniques have evolved, an emerging literature has revealed molecular and functional heterogeneity of lung alveolar mesenchymal cells. In particular, accumulating data demonstrate the previously underappreciated heterogeneity of cells within the Pdgfra+ population of fibroblasts in the adult (Green et al., 2016, Zepp et al., 2017). Single-cell transcriptome analysis combined with spatial distance mapping recently demonstrated that one subpopulation of fibroblasts, defined by the co-expression of Axin2 and Pdgfra, is localized particularly close to AT2 cells and provides signals including Il6, Fgfs, and Bmp antagonists that promote the self-renewal and differentiation of AT2s (Zepp et al., 2017). This fibroblast population has been named the mesenchymal alveolar niche cell or MANC and these cells are thought to have a critical role in homeostatic alveolar regeneration following injury (Zepp et al., 2017).

Immune Response to Alveolar Injury. Resident and circulating leukocytes are thought to also play a critical role in alveolar repair and regeneration, with elegant in vivo and in vitro studies demonstrating that “inflammatory” cytokines have direct effects on the proliferation and differentiation of both airway and alveolar epithelial cells (Danahay et al., 2015, Katsura et al., 2019, Kuperman et al., 2002, Tadokoro et al., 2014, Xie et al., 2018). However, our understanding of the interactions between alveolar epithelial cells and resident or circulating leukocytes is in its infancy. Macrophages, the primary resident immune cell of the alveolus, begin to populate the lung during embryonic lung development (Tan and Krasnow, 2016). LPS stimulation of these early resident macrophages leads to impaired branching morphogenesis, attributed to changes in integrin, Bmp, and Wnt signaling (Blackwell et al., 2011). Whether unstimulated resident macrophages have a normal role in branching morphogenesis or the differentiation of alveolar epithelial cells remains to be elucidated. However, there are multiple lines of evidence indicating that leukocytes play important roles in adult alveolar regeneration, mediated at least in part through bidirectional intercellular communication with alveolar epithelial cells. For example, following either chemical or infectious lung injury, resident alveolar macrophages can stimulate epithelial proliferation through the production of Wnt ligands (Hung et al., 2019). A subset of alveolar macrophages can also act as memory macrophages and help guide the rapid activation of multiple chemokines including those that stimulate neutrophils (Yao et al., 2018).

Compensatory lung growth after partial pneumonectomy provides a relatively simple model to study pro-regenerative epithelial-immune interactions without the confounding effects of infection and inflammation. This model was used to demonstrate that platelets can initiate a regenerative cascade by secreting SDF1 after pneumonectomy. This stimulates capillary endothelial cells to express MMP14, releasing EGF ligands from the extracellular matrix and subsequently promoting AT2 cell proliferation and differentiation (Rafii et al., 2015).

Following partial pneumonectomy, local production of the chemokine CCL2 leads to the recruitment of CCR2+ monocytes to the lung (Lechner et al., 2017). These monocytes and resident macrophages can be polarized by IL13 that is secreted by type 2 innate lymphoid cells, and loss of this axis impairs optimal compensatory lung growth after pneumonectomy. Data from an in vitro co-culture assay suggest that macrophages can directly modulate AT2 cell survival and self-renewal (Lechner et al., 2017). Recruited CCR2+ monocytes have also been implicated in dysplastic alveolar repair following both bleomycin-induced lung injury and other models of lung fibrosis (Misharin et al., 2017, Venosa et al., 2019). These studies highlight the context-dependent roles of CCR2+ monocytes in both normal and abnormal lung regeneration.

In infectious and more destructive lung injury models, it can be difficult to discern the inflammatory roles of leukocytes from regenerative roles, if such a distinction exists. Nevertheless, data from a growing number of contexts have shown that resident and recruited immune cells are essential for resolution of lung injury. For example, the depletion of macrophages during the resolution phase of bleomycin-induced lung injury prolonged the fibrotic response and impaired resolution (Gibbons et al., 2011). This effect was attributed to a decrease in the clearance of accumulated extracellular matrix but could also involve disrupted communication with epithelial progenitor cells or other populations.

Other immune populations are activated or recruited to the alveolar niche following lung injury. We have nascent understandings of the inflammatory cellular diversity and intercellular communications that determine normal versus abnormal lung regeneration in response to lung injury. Understanding this diversity and improving model systems, including more precise animal models and organoid and lung-on-a-chip models that incorporate immune cells, will allow us to study the contributions of immune cell communications that drive lung repair (Gkatzis et al., 2018).

Airway Regeneration

Maintenance and Regeneration of the Proximal Airway Epithelium. The proximal airways in mice and humans are exposed to frequent insults from the environment and serve as the first line of immune and toxin defense in the lung, warming and filtering the air as it passes to more distal regions. Much of what is understood about airway regeneration comes from studies of the mouse trachea, which most closely resembles the structure of human proximal airways. There are many parallels between the murine trachea and the human intrapulmonary airways, with the most relevant for this discussion being the presence of basal cells. In mice, basal cells reside in the trachea and proximal main stem bronchi; however, in humans this population extends for several airway generations (Figures 1 and ​and2 ).2 ). Murine intrapulmonary airways are not pseudostratified and do not contain basal cells which are the primary stem cell population in human airways. Thus, intrapulmonary mouse airways should not be used as a model system for the study of human airways. The pseudostratified upper airway and tracheal epithelium exhibits very slow turnover during health but several of the mature lineages are capable of re-entering the cell cycle to replenish loss of neighboring cells and maintain an epithelial barrier. Basal cells in the proximal airways are the major stem cell population that self-renew and when necessary give rise to multiple cell types such as secretory, goblet, and multi-ciliated cells (Figure 2; Hegab et al., 2012, Hong et al., 2004, Rock et al., 2009, Rock et al., 2011). This process is critical for both maintenance and cellular regeneration after significant injury and is controlled by Notch signaling (Mori et al., 2015, Rock et al., 2011, Ruiz García et al., 2019, Stupnikov et al., 2019). Although it was once thought that basal cells were a rather homogeneous population, recent findings reveal more complexity and have demonstrated their early origins in development (Yang et al., 2018). Careful lineage tracing of cytokeratin 5 (Krt5) basal cells over time indicated that at least two populations of basal cells exist in the upper airway, with one acting more as a self-renewing stem cell, and the other committed to luminal differentiation (Watson et al., 2015). Consistent with this paradigm, depending on enhanced Notch2 signaling or c-myb expression, basal stem cells directly give rise to secretory cells or multi-ciliated cells, respectively (Pardo-Saganta et al., 2015).

Recent studies have uncovered additional complexity within the pseudostratified airway epithelium of mouse trachea and human large airway, with the description of new, rare cell types including CFTR-rich ionocytes (Montoro et al., 2018, Plasschaert et al., 2018). Multiple studies using lineage tracing analysis combined with single-cell transcriptomic work confirmed that the Krt5 basal cell population was capable of giving rise to all the observed cell types within the airway, including the newly identified ionocyte, and other rare epithelial cell subsets such as the tuft cell, which is not normally present in uninjured mouse airways (Montoro et al., 2018, Mori et al., 2015, Rane et al., 2019, Rock et al., 2011, Ruiz García et al., 2019). Moreover, tuft cells, also known as solitary chemosensory cells, ectopically emerge after influenza injury in the mouse and may play a role in post-injury dysplastic remodeling of the lung (Rane et al., 2019). Some of these lineage relationships in mice can be modeled in vitro with primary human cells in culture, highlighting the shared regenerative potential across species (Rock et al., 2009).

While basal cells are a main driver of regeneration after airway injury, other cell types have been shown to contribute as facultative progenitors. Lineage tracing analysis of airway Scgb1a1+ cells revealed that secretory cells proliferate to help maintain the club cell population (Figure 2; Rawlins et al., 2009, Van Keymeulen and Blanpain, 2012) and are a major source of multi-ciliated cells in normal airways, particularly in the more distal murine airways where basal cells are not typically found. In addition, subsets of secretory cells expressing Upk3a in mice, so-called variant-club cells, have been shown to be localized near neuroendocrine bodies, suggesting a possible niche, and are capable of giving rise to both secretory and ciliated cells in development; however, a role in the response to injury is not yet clear (Guha et al., 2012, Guha et al., 2017). Additionally, neuroendocrine (NE) cells also can function as facultative progenitors after airway injury, interact with immune lineages during expansion, and may harbor sublineages with enhanced progenitor capacity in NE bodies (Branchfield et al., 2016, Garg et al., 2019, Ouadah et al., 2019). In extreme situations, mature secretory/club cells (defined by Scgb1a1 expression) have been reported to dedifferentiate in the setting of marked basal cell loss, and contribute to the basal stem cell pool, although the homeostatic or physiologic role of this in injury is not clear (Tata et al., 2013). In addition, a novel pathway of basal cell repletion from the submucosal gland was also reported by two different groups using lineage tracing and multiple airway injury models (Lynch et al., 2018, Tata et al., 2018). These investigators found migration of glandular myoepithelial cells into the airways with subsequent differentiation to mature airway lineages including basal cells.

Regeneration of Distal Airway Epithelium after Injury. A number of distinct, small populations of progenitor cell types have been reported to contribute to regeneration of distal airway epithelia after injury in mice, which lacks basal cells (Barkauskas et al., 2013, Bertoncello and McQualter, 2010, Chen, 2017, Perl et al., 2005). One of the first was a variant club/secretory cell (v-club cells) defined by its location near neuroendocrine bodies and low cytochrome Cyp2f2 expression, making these cells resistant to naphthalene injury and thus a source of regenerative cells after this injury (Giangreco et al., 2009, Hong et al., 2001). More recently Upk3a was identified as a unique marker for v-club cells. A Upk3acreER lineage trace (Guha et al., 2017) demonstrated that these cells give rise to club cells and ciliated cells during homeostasis and after naphthalene airway injury, as predicted from prior studies (Giangreco et al., 2009, Hong et al., 2001, Volckaert et al., 2011). Although seemingly limited, in these studies some v-club/secretory cells were also noted to differentiate into AT2 cells during bleomycin-induced alveolar injury, implying a capacity for mobilization and distal migration.

A second small population of stem/progenitor cells was identified at the branch point between distal murine airways and alveolus and was termed bronchoalveolar stem cells (BASCs). BASCs were initially characterized by dual expression of the secretory cell marker Scgb1a1 and the AT2 marker Sftpc by immunostaining and their localization at the bronchioalveolar duct junction (BADJ) (Kim et al., 2005). Stem cell antigen-1 (Sca1) was also proposed as a marker for these cells and has been used in flow cytometry to purify these cells for in vitro culture studies (Raiser and Kim, 2009). Recently, intersectional-genetic lineage tracing techniques allowed for the specific tracing of BASCs and confirmed that BASCs can generate distal airway club cells after naphthalene injury and peri-junctional AT2 alveolar cells after bleomycin injury or influenza injury (Liu et al., 2019b, Salwig et al., 2019). One of the limitations of these studies is the use of Scgb1a1 expression to mark BASCs, as this gene and protein are known to be expressed in a subset of AT2 cells, which could confound such lineage tracing (Rawlins et al., 2009). While the genetic depletion of the BASC population resulted in a delay of the murine regenerative response to injury, full regeneration was eventually observed, suggesting that BASCs are not absolutely required for lung regeneration.

An additional distal airway stem/progenitor cell population with regenerative potential was identified descriptively as lineage-negative epithelial progenitors (LNEPs), also referred to as distal airway stem cells (DASCs). These cells are distinct from v-club/secretory cells and BASCs. LNEPs were originally defined as distal airway integrin β4+/CD200+ cells without discernible mature lineage markers by protein immunostaining (Vaughan et al., 2015). These cells are also Sox2+, as expected for an airway epithelial cell. Subsequently it became apparent that LNEPs/DASCs are composed of both Trp63+ cells and Trp63− cells. The Trp63+ cells appear to be holdovers from embryonic Trp63+ basal cells and some 20% of these cells could be traced with the Scgb1a1creER mouse line (Yang et al., 2018). Although true basal cells are not normally found in the distal mouse airway, the rare Krt5+/Trp63+ LNEPs/DASCs expand and mobilize after influenza injury to generate collections (or pods) of Krt5+ basal-like cells throughout the heavily damaged areas of influenza-injured mice (Vaughan et al., 2015, Xi et al., 2017, Zuo et al., 2015). This mobilization is initially protective to the mouse but ultimately is a dysplastic response as Krt5+ basal cells have limited potential to differentiate into AT2 cells and the mouse becomes permanently burdened with airway-like cystic structures throughout the alveolar compartment. Appearance of these permanent cystic structures was linked with activated Notch signaling, and blockade of hypoxemia response via deletion of hypoxia inducible factor 1 (HIF1α) in airway cells promotes the contribution of airway cells to regeneration of AT2s and subsequent improvement in oxygen saturation (Xi et al., 2017).

Contribution of Distal Airway Progenitors to Alveolar Repair. Although endogenous AT2s represent the primary regenerative responders to various alveolar insults and regenerate the overwhelming number of new AT2 and AT1 cells after limited alveolar damage, several lines of evidence implicate activation of distal airway epithelial cells as an alternative source of alveolar epithelial cells following certain types of severe lung injury (Barkauskas et al., 2013, Chapman et al., 2011, Xi et al., 2017). Recently, rare MHChigh (H2-K1high) club cell-like progenitors have been described within the larger Scgb1a1 lineage-traced population of all lung cells. These cells have proliferative capacity, appear to give rise to AT2 and AT1 cells following bleomycin-induced lung injury, and can be purified by flow cytometry using anti-H2-K1 antibodies (Kathiriya et al., 2020). H2-K1high cells are a subpopulation of β4/CD200 cells, express low or no mature lung epithelial lineage markers (e.g., Scgb1a1 or Sftpc) at the protein level, and represent ∼5% of all Scgb1a1CreER-labeled lung cells, but exhibit clonogenic potential to generate these broader populations in cell culture outgrowth assays (Kathiriya et al., 2020). H2-K1high cells were found to survive and differentiate into AT2 and AT1 cells in vivo after intra-airway transplantation into bleomycin-injured mice. These cells were also found to be specifically targeted by H1N1 PR8 influenza virus, consistent with a prior report showing β4+/CD200+ cells as a primary target of the viral injury (Quantius et al., 2016). The alveolar epithelial differentiation capacity of H2-K1high progenitors after bleomycin injury contrasts sharply with the differentiation repertoire of the rare Trp63+ cells which predominantly gives rise to dysplastic Krt5+ pods, but virtually no alveolar epithelial cells (Ray et al., 2016, Vaughan et al., 2015, Xi et al., 2017). Additional studies combining injury models with modern cell lineage tracking techniques and single-cell analysis are needed to clarify the injury-specific activation of airway cells and their relative contributions toward alveolar regeneration. Importantly, whether BASCs, LNEPs, and H2-K1high progenitors represent similar or overlapping populations of cells remains unclear. Future studies will need to directly compare these distal airway cells to the resident AT2 and AEP cell populations, to more fully assess their ability to repair and regenerate the alveolar niche after acute injury and in chronic diseases.

Contribution of the Mesenchyme to Airway Regeneration. A series of complex and interconnected interactions are employed to maintain distal airway epithelium during quiescence and after injury within the local niche. The local niche is comprised of the extracellular matrix (ECM) and several mesenchymal cell types. Specifically, the mesenchyme plays a critical role in function of distal airway epithelium by providing a number of signaling cues that ultimately determines the stem cell response during injury. Much of what is known about the interplay in adults between the mesenchyme and the epithelium has been derived from studying lung development. These developmental signaling pathways include Wnt/β-Catenin, fibroblast growth factor 10 and its receptor Fgfr2 (Fgf10/Fgfr), retinoic acid, and sonic hedgehog (Shh) members of transforming growth factor β (Tgfβ) superfamily including bone morphogenetic proteins (Bmp), Hippo/Yes-associated protein (YAP), and Notch signaling. For example, airway smooth muscle cells promote epithelial repair through the production of Fgf10 (Lee et al., 2017, Volckaert et al., 2011) and a population of Pdgfra+ fibroblasts promote the differentiation of multi-ciliated cells through the production of Il6 (Tadokoro et al., 2014). In addition to signaling to the airway epithelium during regeneration, the airway mesenchyme can undergo a phenotypic change following injury that drives abnormal regeneration. A recently identified mesenchymal subpopulation identified by Axin2 expression but lacking Pdgfra expression, the Axin2+ Myogenic Progenitor or AMP, becomes activated after naphthalene airway injury, begins to express Acta2, and contributes the airway fibrosis in the naphthalene model (Zepp et al., 2017). A more thorough review on these pathways and their cell-specific functions can be found elsewhere (Kotton and Morrisey, 2014, Leach and Morrisey, 2018, Lee and Rawlins, 2018, Zepp and Morrisey, 2019).

Human versus Mouse Lung Repair and Regeneration
The human lung contains many structural and anatomic differences which make it unique from its murine counterpart. The murine lung has a 6,000-fold smaller tidal volume, an 8,000 times smaller surface area, and roughly half the generations of airways compared to its human counterpart (Irvin and Bates, 2003, Knust et al., 2009, Thurlbeck, 1967). This significant difference in organ size poses distinct challenges for gas distribution, immune function, and gas exchange. These differences highlight the critical variances that need to be accounted for as the field moves from bench to bedside.

The alveolus is one of the most architecturally conserved regions between the murine and human lung (Figure 1). Conservation of the cellular populations between mouse and human alveoli have been demonstrated through multiple techniques and include the above-described AT1 and AT2 epithelial lineages and the AEP sublineage. Furthermore, the regenerative capacity of the alveolus appears to be retained across species, as discussed above.

The cellular anatomy of trachea and the most proximal airways in mice and the large airways in humans is very similar. The cells of the murine trachea and their progenitor and regeneration capacity appear to be closely aligned with in vitro models of human proximal airway regeneration.

However, the distal conducting airway anatomy of the mouse is quite different from humans. In mice, the intrapulmonary airways are almost completely devoid of cartilaginous rings, bronchial blood support, submucosal glands, and pseudostratified epithelium, all of which is in direct contrast to their human counterparts. Furthermore, murine conducting airways terminate directly into alveolar sacs at the site of the BADJ.

Humans do not have a BADJ, and instead have a distinct distal airway compartment which includes the respiratory bronchioles. These distal respiratory bronchioles are interdigitated with alveolar-like structures that leads into the larger alveolar compartment.

There is no analogous counterpart to the human respiratory bronchioles in mice. Terminal and respiratory bronchioles in humans contain Krt5+ basal cells that are generally absent in the distal airways of mice. To date, a BASC-like cell population has not been found in humans, perhaps due to the lack of the corresponding BADJ anatomical niche.

Thus, the cellular origins of distal airway repair and the origin of airway-based stem/progenitors mobilized during alveolar injury in humans are likely different from that of mice. Given the significant differences between the human and mouse lung, especially in the distal airways, greater focus is needed to study the human lung, both through descriptive assessment using new techniques including single-cell analysis as well as more sophisticated assays including organoids, ex vivo lung explants, and pluripotent stem cell-derived human lung lineages.

Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common adult interstitial lung disease (ILD), a class of pulmonary diseases pathologically defined by interstitial fibrosis, inflammation, or the combination of fibrosis and inflammation (Lederer and Martinez, 2018). A recognized theory of IPF pathogenesis places the initial site of “micro-injury” in the alveolar space with the AT2 cell holding an important position in disease development.

Recent transcriptional interrogation of the distal epithelium in IPF identified activation of cell stress and senescence pathways, and murine modeling of AT2 cell dysfunction from expression of either mutant SFTPC, loss of telomere function, and increased mechanical tension have provided in vivo proof of concept that disruption of AT2 cell homeostasis is a driver of lung fibrosis (Katzen et al., 2019, Naikawadi et al., 2016, Nureki et al., 2018, Reyfman et al., 2019, Wu et al., 2020).

An emerging hypothesis of IPF pathogenesis is that the dysfunctional AT2 cell loses its facultative progenitor capacity creating a regenerative void for lung repair. In support of this hypothesis, a cardinal feature of the pathobiology of IPF is bronchiolization, a term coined decades ago to codify the observation by chest pathologists that epithelial cells with airway markers accumulate in the fibrotic regions of human lungs, appearing to extend the junctional region between distal airways and remaining alveoli (Chilosi et al., 2002).

Micro-honeycomb cysts, another cardinal feature of fibrotic remodeling in humans, are mainly lined by epithelial cells with airway markers, including basal, goblet, and ciliated markers (Seibold et al., 2013). The realization that distal airway stem/progenitors robustly mobilize in mice to occupy alveolar surfaces suggests a parallel process in humans.

While potentially protective against loss of tissue integrity, the progenitors arising from distal airways are also subject to signals that direct their differentiation to airway rather than alveolar phenotypes. These include hypoxia and Notch signaling (Chen, 2017).

Once differentiated to airways cells, differentiation to normal AT2 or AT1 cells may be very difficult and inefficient, potentially accounting for the dysplastic structures that dominate the pathobiology of lung fibrosis (Kumar et al., 2011, Vaughan et al., 2015). In this paradigm, the dysplastic epithelial cysts accumulating progressively in fibrotic human lungs represent remnants of failed repair, as discussed above.

Future efforts to both better understand the development of AT2 cell progenitor dysfunction and to minimize the pathway of airway differentiation of activated progenitors within alveoli of humans could be therapeutic.

Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide, and prevalence continues to increase across the globe with the continued rise in cigarette use and toxic biomasses (Burney et al., 2015). In contrast to IPF, which is characterized by robust cellular production at the site of injury resulting in a dysplastic, fibrotic pulmonary parenchyma, the cardinal feature of COPD in the alveolar space is cellular loss and alveolar simplification, known as emphysema. Mechanistic studies in COPD, however, have failed to generate novel therapeutics aimed at actual lung regeneration.

While much of the work on the deleterious effects of toxin exposures on airway cells has been done in bronchial or upper airway cells, careful anatomic studies have suggested that the site of obstruction is localized to the distal airways and in particular the respiratory airways (McDonough et al., 2011, Koo et al., 2018).

The cellular structure and composition of the distal airway region of the human respiratory system, in particular the respiratory bronchioles, is poorly understood. The final endpoint of emphysema, however, is marked by loss of the alveolar epithelium, which can be studied in murine models. Studies have pointed to an increase in senescence in AT2 cells and the associated endothelium as a mechanism for the development of COPD in certain patient populations and the increased prevalence of the disease with aging (Gao et al., 2017).

Together with the presence of increased senescence in lung fibroblasts from patients with COPD, these studies may suggest an exhausted phenotype in a final common pathway downstream of repeated alveolar repair in the setting of recurring toxic exposure (Müller et al., 2006).

Furthermore, defective alveolar epithelial repair has been associated with reduced Wnt signaling in the COPD microenvironment, and this defective response has been suggested to be downstream of a shift in canonical to noncanonical Wnt signaling in the presence of toxic stimuli such as cigarette smoke (Baarsma et al., 2017).

Whether this change in Wnt signaling is associated with an aberrant or absent response by the Wnt-responsive AEP sublineage in the AT2 cell population, remains unclear. More work is needed to understand how toxic injuries incite distal epithelial cell responses in COPD, in order to be able to develop therapies aimed at bona fide repair of the alveolus, and therefore clinical improvement.

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128675/

---

More information: Finn J. Hawkins et al, Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells, Cell Stem Cell (2020). DOI: 10.1016/j.stem.2020.09.017