Stem Cells Sheltered from Air-Raids Repair Airways

Abstract

Submucosal glands contribute to the luminal secretions of conducting airways in the respiratory tract. In this issue of Cell Stem Cell, both Tata et al. (2018) and Lynch et al. (2018) report that myoepithelial cells of submucosal glands serve as reserve stem cells to regenerate the damaged surface epithelium following severe airway injury.

Submucosal glands (SMGs) are small epithelial appendages composed of ducts and acini embedded in the interstitial layer of conducting airways of the respiratory tract. Their epithelium is populated predominantly by serous and mucous secretory cells and extends continuously to the surface airway epithelium (SAE). SMGs also contain myoepithelial cells (MECs) in the basal side of their acini, which are contractile and expel the SMG secretion into the SAE lumen. This secretion is an essential component of the airway luminal fluid and can be profoundly altered in chronic pulmonary diseases (Liu et al., 2004).

Both the SMG and SAE harbor adult multipotent progenitors for airway regeneration. In the SAE these progenitors are p63+Krt5+ basal cells (BCs), but the progenitor identity in SMGs is less clear due to lack of strict lineage-tracing evidence. SMGs develop postnatally from progenitors in the SAE, and in the adult, they have been proposed to shelter multipotent progenitors from airborne hazardous agents (Anderson et al., 2017). The developmental origin of SMGs from the SAE raises questions about whether the cells in these two compartments migrate out of their own territories and exchange identities to regenerate the damaged epithelium post-injury. Can SMG progenitors repopulate the damaged epithelium? Is there a lineage relationship between the SMG and SAE stem cell compartments?

These questions are collectively addressed in this issue of Cell Stem Cell by Lynch et al. and Tata et al. Both groups independently identify myoepithelial cells (MECs), a population of basal-like alpha-SMA (Acta2)+Krt5+Krt14+ cells in SMGs, as the crucial progenitors for the repair of adult glands. Notably, they provide convincing lineage tracing data supporting the long-held theory that MECs also serve as reserve stem cells for the SAE, recruited from the SMG microenvironment whenever necessary to regenerate both basal and luminal compartments in the SAE. Previous reports using xenograft reconstitution and Krt14-Cre lineage tracing approaches already suggested that SMG cells could differentiate into an SAE fate (Liu et al., 2004; Hegab et al., 2011). Nevertheless, the presence of Krt14+ basal cells under homeostasis rendered this lineage tracing study inconclusive. Moreover, epithelial plasticity as determined by ex vivo xenograft transplantation was questionable for not necessarily reflecting the in vivo scenario, where the endogenous microenvironment is crucial for cell fate restriction.

The study by Lynch et al. provides compelling evidence of the SMG’s participation in injury repair. First, the authors show that the severity of the SAE damage is a key determinant in the recruitment of MECs to the regenerative response. By exposing mice to high doses of naphthalene or sulfur dioxide (SO₂), which leads to extensive death and sloughing of luminal and basal SAE cells, they noticed increased SMG cell proliferation and ectopic Acta2+ cells in the regenerating SAE. By coupling lineage labeling with marker analysis in Acta2-CreERT2 or Myh11-CreERT2 reporter mice to follow the fate of MECs, they revealed a remarkable contribution of SMGs to the SAE. Importantly, this contribution was not observed under milder injury or homeostasis over 1.5 years.

Their spatial-temporal analysis suggested that MEC lineage-labeled cells migrate to the SAE, gradually adopting a local BC identity (Krt5+Krt14+NGFR+) while quenching the MEC markers to then generate luminal descendants (Krt5−Krt8+). Intriguingly, they found that MEC-derived SAE cells still partially retain their original SMG differentiation potential, for example, being unable to generate the secretory club cells characteristic of SAE. The authors reasoned that MEC-derived BCs need additional time to sense the new local cues and fine-tune their differentiation behavior. Indeed, after a second injury, the MEC-derived BCs differentiated more like native SAE tracheal BCs. This mechanism of microenvironmental reprogramming is yet to be explored here and seems reminiscent of that reported in thymic epithelial cells that adopt a hair follicle progenitor fate following epidermal transplantion (Bonfanti et al., 2010). The recruitment of stem cells from an adjacent region to repair a damaged epithelium depleted of its own stem cell pool has also been reported in interfollicular and hair follicle stem cells (Rompolas et al., 2013).

To provide insights into how adult SMGs are recruited for airway regeneration, the authors propose that, similar to SMG morphogenesis, MECs activate Wnt/Lef-1 signaling (Anderson et al., 2017). They then show that mice overexpressing Lef-1 in MECs display an enhanced regenerative response post-naphthalene injury. Intriguingly, they demonstrate that even without injury, simply overexpressing Lef-1 is sufficient to trigger MEC migration and to impart an SAE BC-like signature in MECs. The dominant role of Wnt in the various stem cell pools of the lung has been recently highlighted by the discovery of adult alveolar epithelial progenitors (AEPs) that maintain endogenous Wnt activation (Axin2+) and expand dramatically to regenerate alveoli after acute lung injury (Zacharias et al., 2018; Nabhan et al., 2018).

Employing a different approach, Tata et al. (2018) arrive at observations similar to that of Lynch et al. (2018). The authors performed single-cell RNA-seq in regenerating airways post-naphthalene and found SAE cells expressing SMG markers, such as Acta2 and the SRY-box gene Sox9. Interestingly, Sox9 is known to mark multiple progenitors of the developing lung, including distal buds and the primordial placodes in SAE that give rise to the SMGs (Rockish et al., 2013; Anderson et al., 2017). Notably, Tata et al. found that in uninjured adult mice, Sox9 labels all SMG cells but is absent in the SAE. Using Sox9-CreER mice, they revealed no SMG contribution to the SAE under homeostasis but found extensive SAE labeling in naphthalene-injured animals. They mechanistically implicate Sox9 in controlling the migratory and proliferative behavior of SMG-derived cells in regeneration. Knocking out Sox9 conditionally in MECs and SAE BCs with a Krt5-CreER line severely impaired the expansion and ectopic appearance of SMG-derived cells in the regenerating SAE.

How robust are these MEC-derived basal cells as progenitors? The authors addressed this question by engrafting MECs isolated from Acta2-CreER; R26-tdTomato mice onto denuded tracheal scaffolds. Cultured explants confirmed the ability of these progenitors to give rise to basal (Krt5+) and SAE luminal (secretory, multiciliated) cell types. Lastly, Tata and colleagues provide initial evidence of potential evolutionary conservation in these events, showing SMG contribution to SAE repair in injured porcine airways. The recognition of differences in injury response, such as the presence of Sox9+Acta2− ectopic cells in regenerating SAE, warrants further extensive study so that we may better understand the contribution of SMG to airway repair in large animals and ultimately in humans.

What new and unresolved questions emerge from these studies? Important areas for future research include the understanding of key compartment-specific differences in mesenchymal niches and epigenetic changes involved in the SMG-SAE phenotype switch. Better defining the reported role of Lef-1 using loss-of-function approaches and finding how Lef-1 levels balance self-renewal and differentiation in SMGs is equally important. Furthermore, identification of the Sox9 downstream targets regulating the MEC-SMG cell behavior should uncover new mechanistic insights into the remarkable observations reported here. Finally, it would be interesting to learn how continued inflammatory changes in the SMG milieu in severe chronic respiratory infections influence the SMG-SAE repair mechanism.