Repairing Mechanisms for Distal Airway Injuries and Related Targeted Therapeutics for Chronic Lung Diseases

Abstract

Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), involve progressive and irreversible destruction and pathogenic remodeling of airways and have become the leading health care burden worldwide. Pulmonary tissue has extensive capacities to launch injury-responsive repairing programs (IRRPs) to replace the damaged or dead cells upon acute lung injuries. However, the IRRPs are frequently compromised in chronic lung diseases. In this review, we aim to provide an overview of somatic stem cell subpopulations within distal airway epithelium and the underlying mechanisms mediating their self-renewal and trans-differentiation under both physiological and pathological circumstances. We also compared the differences between humans and mice on distal airway structure and stem cell composition. At last, we reviewed the current status and future directions for the development of targeted therapeutics on defective distal airway regeneration and repairment in chronic lung diseases.

Keywords

lung stem cell cell niche chronic lung diseases

Introduction

The lung tissue had been considered highly quiescent with limited regenerative capacities; however, large body of evidences has revealed that the lung tissue possesses remarkable reparative capacities responsive to varieties of distal airway injuries¹. The distal airway comprises terminal bronchioles (TB) and alveoli that perform essential respiratory functions. The bronchioles branch off from a hierarchy of bronchi that are stemmed from the trachea, and the alveoli are the basic units for gas exchange. The distal airway epithelium is composed of a wide variety of epithelial cells^2,3, several of which have been reported as somatic stem cells (SSCs) (Fig. 1).

Figure 1.

The anatomical structures of human and mouse airways and their epithelial components. TB: terminal bronchioles; BADJ: bronchioalveolar duct junctions; BASC: bronchioalveolar stem cells; AT1: alveolar type I; AT2: alveolar type II; RAS: respiratory airway secretory; RB: respiratory bronchioles.

Different SSCs have their own unique niches, including SSCs and their progenies, but also a variety of heterogeneous cell types and extracellular matrix (ECM). Under physiological conditions, SSCs are largely in dormant state, maintaining low level of self-renewal and differentiation. Once the tissue is injured, the SSCs receive signals from surrounding microenvironment that subsequently drive the regeneration or reparative process. However, the reparative capacities of SSCs are frequently compromised if the niche environment is not supportive⁴.

Human and mouse lungs have similar histological structure. The lung is composed of trachea, bronchi, bronchioles, and alveoli from the proximal to the distal airways. Trachea and bronchi are mainly composed of club cells, goblet cells, multiciliated cells, basal cells, and a small number of neuroendocrine cells, brush cells, and ionocytes. Unlike human, mouse small airways do not contain basal cells or goblet cells. The human distal airway is connected to the alveolar through a transitional airway region (also known as the respiratory bronchiole [RB]). In this region, there are rare newly discovered stem cells called respiratory airway secretory (RAS) cells⁵. There are no RB in mice, which directly transit to the alveolar area through the middle airway. The distal bronchioalveolar duct junction (BADJ) region in the mouse lung contains bronchioalveolar stem cell (BASC) population, whose counterparts have not been found in human lung. Both mouse and human alveoli contain two cell populations, alveolar type II (AT2) and alveolar type I (AT1) cells (Table 1).

Table 1.

Identification of Pulmonary Epithelial Stem Cells.

Cell type	Location	Makers	Differentiation repertoire	References
Basal cells	Airways	TRP63, KRT5, NGFR, PDPN	club, ciliated, AT2s	^7,23
Club cells	Airways	SCGB1A1^high, SCGB3A1, SCGB3A2	ciliated, basal, AT2s	^16,17
Variant club cells	NEBs in bronchioles	SCGB1A1, UPK3A	club, ciliated	^19,21
Distal airway stem cells (DASCs)	Distal airways (bronchioles and alveolar regions)	TP63, KRT5	Krt5⁺ “pods”	²³(p. 1),²⁴
Bronchioalveolar stem cells (BASCs)	Bronchioalveolar-duct junctions (mouse)	SFTPC, SCGB1A1	club, AT2s	²⁸
Respiratory airway secretory cells (RASCs)	Respiratory bronchioles (human)	SCGB3A2^high, SCGB1A1	AT1s	⁵
Alveolar type 1 cells (AT1s)	Alveolus	AQP5, PDPN, HOPX, RAGE,	AT2s	^38,42
Alveolar type 2 cells (AT2s)	Alveolus	SFTPC, LAMP3	AT1s	^36,39

Distal Airway SSCs

SSCs in the Bronchi

Basal cells

Human lung basal cells are distributed throughout the main airways to the terminal and respiratory bronchioles (TRB), and owing to the lack of counterpart RB, the mouse basal cells are mainly distributed in the main bronchi only⁶. Basal cells occupy ~6% of the epithelium in the smallest conducting airways and are commonly identified as KRT5⁺TP63⁺ at steady state⁷. Lineage-tracing experiments validated that Krt14⁺ basal cells are multipotent airway progenitors that could give rise to ciliated cells and secretory cell types following naphthalene-induced lung injury (Box 1)^8,9. In vitro sphere-forming assays (Box 2) demonstrated that both human and mouse airway basal cells can self-renew and generate luminal cells⁷. Gene expression analysis and histomorphometric studies with both steady-state and naphthalene-injured mouse trachea have demonstrated that there are two basal cell pools (Krt14⁺ and Krt14⁻) that restore the tracheobronchial epithelium coordinately^10,11. More recently, a study pointed out that NOTCH signaling is required for the differentiation, but not self-renewal of basal cells^12,13. Another study showed that immediately following injury, one p63⁺ basal cell population activates intracellular NOTCH2 signaling and generates secretory cells directly, while the other basal cell population expresses c-Myb and directly yields ciliated cells¹⁴. In addition, a rare population of SOX9⁺/P63⁺/KRT5⁺ basal cells has been shown to be able to regenerate human alveoli¹⁵.

Box 1.

Models of lung injuries.

1. Pneumonectomy (PNX)

Pneumonectomy is an animal model of alveolar regeneration induced by pneumonectomy^135,136. In different species, studies have found that after surgical removal of one or more lobes, compensatory regeneration of the remaining lung tissue induces the formation of new blood vessels, epithelial and mesenchymal cells, and alveolar septum to restore alveolar surface area. Eventually, alveolar stem cells proliferate and differentiate, new pulmonary alveoli regenerate, respiratory function recovers, and respiratory tension in the alveolar area returns to normal physiological levels^40,42,62,71.

2. Bleomycin (BLM)

Bleomycin is an anti-cancer drug. One of the side effects is pulmonary fibrosis. Most studies suggest that the mechanism of BLM-induced pulmonary fibrosis is to induce DNA breaks, produce free radicals, induce oxidative stress, cause cell apoptosis or necrosis, leading to pulmonary inflammation and fibrosis^137,138. At present, bleomycin-induced pulmonary fibrosis animal model is the most widely used experimental model for pulmonary fibrosis^138,139.

3. Naphthalene

Naphthalene is an aromatic hydrocarbon that is used to induce ablation of club cells¹⁸. After naphthalene-induced damage, cytochrome P450-2F2 (CYP-2F2) metabolizes naphthalene to toxic epoxides, and club cells expressing cytochrome P450-2F2 (CYP-2F2) are selectively ablated^19,20. High doses of naphthalene can cause epithelial denuding damage and even lead to loss of surface basal cells^140,141.

4. Elastase

In emphysema, elastase is involved in alveolar wall degradation. Studies have shown that exogenous administration of elastase can cause emphysema¹⁴². The most common method of inducing emphysema is intratracheal instillation of porcine pancreatic elastase (PPE) or human neutrophil elastase (HNE)¹⁴². Intratracheal instillation of large amounts of elastase in mice can lead to alveolar destruction and enlarged air space. Protease-induced emphysema animal model is a commonly used as mature lung injury model at short time expense¹²².

Box 2.

Lung Organoids Culture Models.

1. Airway-liquid interface (ALI)

ALI uses primary bronchial cells or small airway epithelial cells to establish multi-layered mucosal ciliated epithelium to simulate human respiratory epithelium^143,144. In ALI, the basal side of the cell was in contact with the liquid medium, while the apical side of the cell was exposed to air. ALI are often used to identify growth and differentiation factors and to screen drugs for respiratory diseases¹⁴³.

2. Organoids from basal cells

Based on the source of basal cells, organoids of basal cells can be divided into two categories. The organoids derived from tracheal basal cells are called the tracheospheres, while those derived from bronchi or airways are called the bronchospheres¹⁴⁵. Tracheospheres from mouse tracheal basal cells formed spheres with visible lumens within two weeks in Matrigel, with basal cells in the outer layer and club and ciliated cells in the inner layer⁴. Bronchospheres from human bronchial basal cells are more complicated than those from mice and consist of basal cells, club cells, goblet cells and ciliated cells¹⁴⁶.

3. Organoids from club cells

In mouse distal airways, when co-cultured with mesenchymal cells or mouse lung endothelial cells, club cell-derived organoids produce three different types of colonies, including bronchioles, alveoli and bronchioloalveolar colonies⁸⁷.

4. Organoids from alveolar cells

Primary AT2 cells digested and isolated from donor lungs can form alveolar organoids when co-cultured with feeder cells in matrigel¹⁴⁷. The organoids from AT2 cells are composed of two types of epithelial cells. The polarized AT2 cells are on the outside with their basal membrane facing the Matrigel, while the AT1 cells fold toward the inside⁴¹. Recent studies have shown that alveolar organoids in mice and humans can be cultured without feeder cells using chemical media containing factors related to lung development¹⁴⁸.

5. Organoids from pluripotent stem cells

Lung organoids can also be derived from pluripotent stem cells such as embryonic stem cells and iPSCs^149,150.

The PSC-derived lung cells retain the potential to differentiate into each cell type of body. The structure of human lung organoids produced by pluripotent stem cells is similar to that of primary lung, which is composed of proximal airway epithelium, distal alveolar epithelium and other cell types¹⁴⁹.

AT2: alveolar type II; AT1: alveolar type I; iPSCs: induced pluripotent stem cells.

Club cells

With specific expression of the secretoglobins Scgb1a1 and Scgb3a2, club cells are known as predominant progenitors for the maintaining and repair of bronchial epithelium. Genetic lineage tracing study demonstrated that Scgb1a1⁺club cells can self-renew and differentiate into ciliated cells¹⁶. Of note, studies of dioxide-induced injury and influenza viral infection in mice demonstrated that club cells can also dedifferentiate into basal cells when basal cells are ablated¹⁷. However, it remains unclear whether this process also occurs in physiological condition.

Variant club cells

Naphthalene is an aromatic hydrocarbon that is used to induce the ablation of club cells¹⁸. A pollutant-resistant subpopulation of club cells called variant club cells expressing Clara cell secretory protein (CCSP/SCGB1A1) and Upk3a were identified in a study of naphthalene-induced club cell ablation^19,20. This variant club cell subpopulation lacks the expression of cytochrome P450-2F2 (CYP-2F2), which generates highly cytotoxic 1R, 2S-naphthalene epoxide^19,20. Variant club cells normally locate around neuroendocrine bodies (NEBs) and within the BADJs, and these cells can replenish secretory and ciliated cells under both physiological and pathological (club cell ablation) conditions. In addition, variant club cells can also give rise to AT1 and AT2 cells after bleomycin-induced alveolar injury^21,22.

Distal airway stem cells (DASCs)

Emerging number of reports suggest that alternative subsets of distal airway epithelial cells may serve as facultative progenitors and promote regeneration of injured alveolar epithelium. Lineage tracing study revealed that Trp63⁺stem cells originated from the bronchiolar epithelium can be detected in alveolar regions and assemble into Krt5⁺ pods following pulmonary infection of H1N1 influenza virus²³. Kumar et al.²³ defined this facultative progenitor cells as DASCs. Controversially, earlier studies have shown that DASCs can differentiate into AT1, AT2 cells as well as bronchiolar secretory cells following H1N1 infection²⁴. However, subsequent articles showed that DASCs are unable to repair alveolar epithelium after influenza virus infection in mice^25,26.

Bronchioalveolar stem cells

At the BADJs in the mouse, a rare population of stem cells expressing markers of both club cells (Scgb1a1) and AT2 cells (Sftpc) was called BASCs. BASCs are resistant to bronchiolar and alveolar damage and proliferate during epithelium restoration^27–30. However the role of BASCs during lung repair and regeneration in vivo remains controversial: Rawlins et al.¹⁶ concluded that BASCs do not contribute into postnatal maintaining of bronchioles and alveoli; in contrast, recent two studies demonstrated that BASCs could generate the majority of distal lung airway cells including club cells, ciliated cells, AT1, and AT2 cells after bronchiolar damages and rarely contribute to cellular turnover under homeostatic conditions by using lineage-tracing system^31,32. However, the structure of BADJ has not been found in the human lung. Therefore, it is important to confirm whether BASCs or similar cell subsets exist in human lung.

RB cells

Human distal airways contain a unique structure of RB that are absent in mice⁶. RB are frequently injured in several chronic human respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF)³³. Since the mouse lung lacks the structure of RB, it remains one of the least known regions of the human lung. Surprisingly, two back-to-back papers recently revealed that a new lineage of stem cells is present in this mysterious region of humans distal airways^5,34. Morrisey et al. identified a distinct population of RAS cells expressing high SCGB3A2 level. Results of organoid model (Box 2) showed that these RAS cells are unidirectional progenitor cells that can rapidly differentiate into AT2 cells, a process controlled by coordinated NOTCH and Wnt signaling⁵. Using spatial transcriptomics and single-cell profiling of distal airways, Tata et al. identified several novel TRB cell types, including airway-associated LGR5⁺ fibroblasts, TRB-specific alveolar type-0 (AT0) cells, and TRB secretory cells (TRB-SCs). They showed a new differentiation program that AT2 cells transiently acquire an AT0 state from which they can differentiate into either AT1 cells or TRB-SCs. Although more research is needed to unveil how the RB structures and related stem cells impact human lung function and regeneration, these two articles have milestone significance in the field of human alveolar research.

Alveolar SSCs

Pulmonary gas exchange occurs in alveolar sacs lined by two epithelial cell types: AT2 and AT1. AT1 cells are long flat squamous cells covering majority of the gas-exchange alveolar surface, while AT2 cells are small, cuboidal cells secreting surfactants that prevent alveolar collapse^18,19. AT2 cells have been reported as SSCs^35,36. Recently, it has been reported that AT1 cells also have stem cell plasticity during alveolar development and regeneration^37,38 (Fig. 2).

Figure 2.

Trans-differentiation network among somatic stem cells in distal airways. BASC: bronchioalveolar stem cells; AT2: alveolar type II; RAS: respiratory airway secretory; AT1: alveolar type I.

AT2

AT2 cells are the most thoroughly studied alveolar regenerative progenitors to date. After acute lung injury induced by NO₂ exposure in rats, AT2 cells can self-renew and differentiate into AT1 cells^35,36. These data support that AT2 cells are the progenitor cells for AT1 cells in rodents³⁹. Moreover, a medical study first provided evidence that new lung growth occurred in an adult human after a right-sided pneumonectomy (PNX) (Box 1)⁴⁰. Genetic lineage-tracing experiments showed that AT2 cells self-renew and differentiate at a low rate over 1 year. Moreover, when the lung epithelial cells are ablated after injury, the remaining AT2 cells can proliferate rapidly⁴¹. More details on AT2 functioning at the alveolar stem cells will be reviewed in the following section.

AT1

AT1 cells have long been considered as terminally differentiated cells. However, recent studies have shown that AT1 cells also possess stem cell plasticity. In 2015, Jain et al.⁴² discovered that Hopx⁺ AT1 cells proliferated and dedifferentiated into AT2 cells during adult alveolar regeneration after PNX (Box 1), and this process was regulated by tumor growth factor-beta (TGF-β) signaling. Moreover, a subsequent study further explored the changes of AT1 cells at the transcriptome level and found that there are two types of postnatal AT1 cells: Igfbp2⁺ Hopx⁺ and Igfbp2⁻ Hopx⁺ AT1 cells. The findings of this study supported that there is an “immature” period of Igfbp2⁻ Hopx⁺ AT1 cells during the process of AT2-to-AT1 differentiation in alveolar regeneration. The stem cell like “immature” Igfbp2⁻ Hopx⁺ AT1 cells are bidirectional and capable to trans-differentiate into AT2 cells under pathological conditions, while Igfbp2⁺ Hopx⁺ AT1 cells represent the terminally differentiated population of AT1 cells ³⁸. In the latest study, AT1 cells can be reprogrammed into AT2 cells to promote alveolar regeneration after neonatal injury, and this plasticity is retained during alveolar maturation, while the ability of AT2 cells to differentiate into AT1 cells is restricted in the adult mouse lung³⁷. Up to now, understanding of AT1 cell development and heterogeneity is very limited in the field of lung regeneration. Future investigations are needed to validate whether the reported AT1 plasticity can be extrapolated to other species or other lung injury models.

Basal cells, club cells, and AT2s are the main SSCs in distal airways. SSCs transdifferentiate into each other to maintain lung homeostasis under physiological and pathological states.

Repairing Mechanisms for Distal Airways

Acute pulmonary injuries rapidly induce the intrinsic repairing programs of the SSCs to restore the structural and functional homeostasis. In general, majority of our knowledge for repairing mechanisms of distal airways were obtained from studies of acute pulmonary injuries, during which SSC-intrinsic and -extrinsic factors work orchestrally to facilitate balanced self-renewal and differentiation of SSCs.

Alveolar Repairing and Regeneration

Transitional AT2 states and related regulatory signaling pathways during alveolar regeneration

In the alveolar epithelium, only small proportions (~20%) of AT2 are alveolar epithelial progenitor (AEP) cells that are quiet stable during physiological homeostasis⁴³. When acute lung injuries occur, the AEP pool expands rapidly to regenerate the damaged alveolar epithelium by replenishing large number of AT2 and AT1; however, the AEP pool shrinks back to physiological state once the repairing process is accomplished^35,43. The single-cell omics technique has characterized three transitional states of AT2 during the alveolar repairing process: self-renew (proliferating), self-renew cessation (cell cycle arrest), and transdifferentiating (AT2-to-1)⁴⁴. Several groups have coincidentally defined a AT2 state as pre-AT1 transitional state (PATS) or damage-associated transient progenitor (DATP) cells, which shares the similar transcriptomic features as AT1, but its expressions of canonical AT1 markers including Pdpn, Hopx, and Cav-1 are much lower^45,46–48.

Numerous molecular and genetic studies have identified multiple pathways involved in the regulation of alveolar regeneration, including Wnt, FGF, BMP, NOTCH ligands, TGF-β and retinoid acid (RA). Synergistic activation of these signaling pathways occur dynamically and orderly at different stages of the alveolar repair process. The Wnt/β-catenin, FGF/FGFR2 and ETS variant transcription factor 5 (Etv5) signaling facilitate AT2 self-renew but inhibit AT2-to-1 trans-differentiation^35,43,49,50, while BMP signaling promotes AT2-to-1 transdifferentiation⁵¹. For TGF-β signaling, it was initially considered that the autocrine TGF-β1/Smad pathway is important for AT2-to-1 transdifferentiation⁵², further study precisely revealed that TGF-β signaling needs to be temporally upregulated for cell cycle arrest in AT2, but downregulated afterwards for AT2-to-1 trans-differentiation⁴⁴. The NOTCH pathway is activated in AT2 for AT2 proliferation in response to acute injuries, while subsequent expression of Dlk1 is required to temporally modulate NOTCH signaling for AT2-to-1 transition⁵². RA indirectly promote alveolar repair by rejuvenating regenerative fibroblasts⁵³.

Alveolar microenvironment fuels alveolar repair

Wide variety of components in the alveolar niches, such as fibroblasts, endothelial and immune cells, produce and secrete supporting factors such as Wnt ligands, FGF, BMP, NOTCH ligands, TGF-β, RAs, and so on, to cultivate the AEPs to launch the repairing programs in response to injuries (Fig. 3).

Figure 3.

Reparative niche for alveolar regeneration: (A) Pdgfrα⁺ fibroblasts maintain the stemness of AT2 cells by secreting short-range Wnt ligands. PCECs regulate epithelial homeostasis by modulating the expression of HGF and EGF. Macrophages promote alveolar repair by producing inflammatory cytokines (IL-1β and TNF-α) and HGF. Damaged AT2s secrete the “alarmin” molecules IL-33 that in turn activates ILC2s to release IL-13 and IL-5, which indirectly facilitate AT2 proliferation by promoting differentiation of alternately activated macrophages. (B) A transitional AT2-to-1 state exists during alveolar regeneration. Persistence of aberrant AT2-to-1 transient cell state leads to impaired alveolar regeneration. AT2: alveolar type II; PCECs: pulmonary capillary endothelial cells; HGF: hepatocyte growth factor; EGF: epidermal growth factor; IL: Interleukin; TNF-α: tumor necrosis factor alpha; AT1: alveolar type I.

Fibroblasts serve as the main source of the Wnt ligands

Three fibroblast subpopulations are present in the alveoli: retinol-storing Pdgfrα⁺ lipofibroblasts, cytokine-producing stromal fibroblasts, and mechanically contractile myofibroblasts^54–57. The AEPs are located near the single Pdgfrα⁺ lipofibroblast niches, which are essential for alveolar development by secreting short-range Wnt ligands to facilitate AT2 stemness maintaining and also producing RAs and lipids to promote surfactant synthesis in AT2^35,54–57. Persistent or repeated fibroblast activation leads to pathogenic airway remodeling and impaired capacity to produce supporting factors for SSCs regeneration under chronic pathogenic conditions^54,58.

Pulmonary capillary endothelial cells promote lung alveologenesis

Growing evidence has proved that epithelial-vascular interactions are essential for tissue patterning^59,60. Alveolar epithelial cells are surrounded by pulmonary capillary endothelial cells (PCECs)⁶¹. PCECs are not just passive conduits for the delivery of oxygen, but also promote alveologenesis by providing angiocrine growth signals^59,60. Unilateral PNX induce VEGFR2 and FGFR1 activation within PCECs of the remaining lung lobes, leading to increased production of matrix metalloprotease MMP14, which subsequently promotes regenerative alveolarization by releasing epidermal growth factor (EGF)-like ligands⁶². Pulmonary capillaries regulate epithelial morphogenesis by modulating the expression of hepatocyte growth factor (HGF), which is necessary for distal airway development⁶⁰. Besides, endothelial cell derived HGF also indirectly facilitating lung regeneration through suppressing tissue fibrosis by preventing aberrant activation of the NADPH Oxidase 4 (NOX4) in perivascular fibroblasts^63–65. Recent articles have shown that the Car4⁺PCECs receive reparative signals from adjacent AT1 cells during influenza infection induced alveolar revascularization^66,67.

Immune niches for alveolar epithelium cells

Macrophages/monocytes eliminate foreign substances by engulfing pathogens and triggering immune response^68,69. Growing evidence has shown that macrophages and bone marrow-derived monocytes contribute into acute injury-induced airway epithelium regeneration^70–72. Mechanistically, macrophages promote alveolar repair by producing inflammatory cytokines (IL-1β and TNF-α) and growth factors (HGF)^70,73–75. Damaged AT2s secrete the “alarmin” IL-33, which in turn activates group 2 innate lymphoid cells (ILC2s) that release IL-13 and IL-5. The aforementioned type II cytokines indirectly facilitate AT2 expansion by promoting differentiation of alternatively activated macrophages^71,76. Controversially, another study reported that IL-13 disrupts AT2 stem cell activity⁷². Future studies are important for dissecting other immune cell subpopulations that mediate alveolar regeneration.

Regeneration of Bronchi Epithelium

The distal airways mainly contain two types of SSCs, basal cells and club cells, which function cooperatively to maintain distal airway homeostasis and regeneration^8,9,16 (Fig. 4).

Figure 4.

Reparative niche for bronchi epithelium regeneration. MCs: mesenchymal cells; ASMCs: airway smooth muscle cells; IL: interleukin.

NOTCH and TGF-β/BMP/SMAD signals promote basal cell differentiation and pseudostratified epithelium regeneration

Adult KRT5⁺ basal stem cells turnover slowly to maintain airway epithelial integrity during homeostasis⁷⁷. When the airway epithelium is injured, basal cells proliferate rapidly and differentiate into all cell types of pseudostratified epithelium to repair the damaged airways^14,78. Fate determination of basal cell differentiation is controlled by NOTCH signaling pathway. Basal cells tend to differentiate into club cells when NOTCH2 signal is active; otherwise, they differentiate into ciliated cells¹⁴. In addition, activation of the TGF-β/BMP/SMAD signaling pathway promotes basal cell differentiation and luminal regeneration after injury, while inhibition of this signal leads to basal cell hyperplasia⁷⁹.

BMP and NOTCH signals are essential for club stem cell self-renewal

Club cells mainly exist in the noncartilaginous region of the distal airway, especially in the bronchioles. Club cells can self-renew and differentiate into ciliated cells, making club cells the main progenitors for the maintenance and repair of bronchial epithelium¹⁶. BMP and NOTCH are involved in the self-renewal and differentiation of club stem cells. Bmpr1a-mediated nonclassical BMP signaling pathway is essential for the expansion of regenerating club cells⁸⁰. Loss of Bmpr1a, Tak1, or Mapk14 in adult mouse club cells leads to defective bronchial regeneration and repair. Activation of NOTCH signaling can maintain club cell fate and prevent differentiation of club cells into ciliated cells^81,82. p53 deficiency promoted club cell self-renewal⁸³.

Mesenchymal stromal cells regulate bronchi epithelium regeneration

Mesenchymal cells (MCs), in proximity to the SSCs in the distal airways, have been shown to regulate the stemness and differentiation of SSCs through FGF, Hippo, Wnt, NOTCH signaling pathways. Under steady state, MCs maintain basal stem cell status in cartilaginous airways by providing FGF10 to basal cells to inhibit Hippo signal (increase nuclear yap proteins)^84,85, while upon airway injuries, increased expression of FGF10 in airway smooth muscle cells (ASMCs) and Wnt7b in airway epithelium allow basal cells to move into noncartilage-conducted airways for epithelial regeneration⁸⁴. The Wnt ligands produced by MCs do not contribute much to basal cell proliferation under physiological condition but are essential for epithelium repair post injuries⁵⁴. Following pulmonary injuries, MCs produce Wnt ligands to promote proliferation of basal cells, which further secrete Wnt ligands and subsequently regulate ciliated cells generation from basal cells⁵⁴. Similarly, upon airway injury, IL-6 produced by MCs activates STAT3 in basal stem cells, which in turn inhibits NOTCH signaling and promotes basal stem cell differentiation into ciliated cells⁸⁶. LGR6⁺ ASMCs in bronchiolar epithelium can also help club stem cells repair damaged airway epithelium through Wnt-FGF10 signaling⁸⁷. Hedgehog signaling in Scgb1a1⁺ club cells of the distal airways control postnatal lung mesenchyme quiescence and regulates distal airway epithelium homeostasis and regeneration⁸⁸.

MCs maintain basal stem cell status in cartilaginous airways by providing FGF10 to basal cells to inhibit Hippo signal, while upon airway injuries, increased expression of FGF10 in ASMCs and Wnt7b in airway epithelium allow basal cells to move into noncartilage-conducted airways for epithelial regeneration. IL-6 produced by MCs activates STAT3 in basal stem cells, which in turn inhibits NOTCH signaling and promotes basal stem cell differentiation into ciliated cells.

Impaired Injury-Induced Reparative Programs of SSCs in Chronic Lung Diseases

Chronic lung diseases, such as COPD and IPF, involve long-term and irreversible airway destructions and pathogenic airway remodeling, leading to dampened capacities of lung tissue to launch the “injury-induced reparative programs”⁸⁹; however, the underlying mechanisms remain largely unknown. In most cases, the only treatment for chronic lung disease is tissue transplantation, and the 5-year survival rate post transplantation is only 59%⁹⁰. Understanding of causal mechanisms responsible for the impaired reparative programs is of great significance for the development of targeted therapeutics for chronic lung diseases. Currently speaking, it is suggested that the airway SSCs under repeated injury-repair cycles during the development of chronic lung diseases possess exhausted SSCs stemness caused by SSC-intrinsic and extrinsic mechanisms^91,92.

SSC-Intrinsic

Accumulating evidence suggests AT2 cell injury is an early event in the development of chronic lung diseases^93,94. The common features of COPD and IPF are great loss of functional AT2s accompanied with increased AT2s aging and senescence^89,95–98. Genetic studies demonstrate that senescence of AT2s is sufficient to initiate progressive IPF-like pathologies⁹⁹. Abnormal telomerase expression and misfolded surfactant proteins within AT2s are also closely associated with AT2s senescence and apoptosis^100,101. Therefore, elucidating the underlying mechanisms of AT2s senescence is essential for the development of effective therapies for alveolar repair.

Recent studies have shown that persistence of aberrant AT2-to-1 transient cell state controlled by TP53 signaling fails to repair damaged alveoli and plays important roles in the pathologies of IPF^45,46. The AT2-to-1 transitional cells are trapped in the intermediate stage of abnormal differentiation and are incapable to terminally differentiate into AT1 cells in IPF, leading to destroyed alveolar repairment^45,46. The aberrant PATs are characterized by enriched p53 and TGF-β signaling, cell senescence and DNA damage⁴⁵.

SSC-Extrinsic

Functional reparative capacities of SSCs need permissive niche environment, which is frequently compromised in chronic lung diseases. Progress of lung tissue fibrosis and airway remodeling are accompanied with activation of mesenchymal stromal cells and dysregulated lung endothelial cells, which are incapable of producing enough growth factors and cytokines (HGF, FGF7. . .) to facilitate distal airway regeneration and repairment^102–104. Zepp et al.⁵⁴ identified two MC subpopulations in mouse lung tissue: one is mesenchymal alveolar niche cells (MANCs), which secrete FGF7 to promote AT2s regeneration and differentiation upon lung injury; the other is Axin2⁺ myofibrogenic progenitors (AMPs), which tend to proliferate uncontrollably to form fibrotic scars. Lee et al.¹⁰² reported that decreased expression of the thrombospondin 1 (TSP1) by lung endothelial cells suppresses distal airway regeneration.

Therapeutic Strategies for Distal Airway Repair in Chronic Lung Diseases

Development of targeted therapeutics for distal airway regeneration and repairment in chronic lung diseases has been very challenging, and most of the attempts are preliminary and pre-clinical. The general rationale is to rejuvenate SSCs stemness or/and restore the reparative niche environments (Tables 2 and 3, Fig. 5).

Table 2.

Stem Cell Therapy in Preclinical Trials.

Stem cell type	Source	Model	References
AT2s	Rat	Rat, BLM (intratracheal instillation)	¹²⁵
iPSCs	human	Rat, BLM (intratracheal instillation)	¹²⁶
MSCs	Rat	Rat, LPS (intratracheal instillation) + CS-induced	¹²⁷
ESCs	human	Mouse, BLM (intratracheal instillation)	¹²⁸

AT2s: alveolar type II cells; BLM: bleomycin; iPSCs: induced pluripotent stem cells; MSCs: mesenchymal stem cells; ESCs: embryonic stem cells.

Table 3.

Stem Cell Therapy in Clinical Trials.

ClinicalTrials.gov ID	Status	Conditions	Stem cell type	Interventions
NCT00683722	Phase II (completed)	COPD	MSCs	400× 10⁶ cells, intravenous infusions on Days 0, 30, 60, and 90
NCT04047810	Phase I (active, not recruiting)	COPD	MSCs	0.5 to 2 × 10⁶ cells/kg, intravenously once
NCT02745184	Phase II (recruiting)	IPF	LSCs	1 × 10⁶ cells/kg, fiberoptic bronchoscopy
NCT01306513	Phase I (recruiting)	Emphysema	MSCs	Intravenous

COPD: chronic obstructive pulmonary disease; MSCs: mesenchymal stem cells; IPF: idiopathic pulmonary fibrosis; LSCs: lung stem cells.

Figure 5.

Overview of current therapeutic strategies for distal airway repair in chronic lung diseases. SSCs: somatic stem cells; DQ: dasatinib and quercetin; AAV: adeno-associated virus; iPSCs: induced pluripotent stem cells; hESCs: human embryonic stem cells; MSCs: mesenchymal stem cells; LSCs: lung stem cells.

Therapeutic Interventions on SSCs

Antisenescence

SSCs in the distal airways of chronic lung diseases often express aging-related indicators^89,95,96,105, making anti-senescence strategy an attractive option to rescue SSCs stemness. Delivery of telomerase by adeno-associated virus (AAVs) vectors in AT2s increased telomere length and restored regenerative capacity of AT2s in mice with pulmonary fibrosis¹⁰⁶. Senolytic drugs such as dasatinib and quercetin have been shown to delay the progression of pulmonary fibrosis in aged mice and reduce the aging of alveolar epithelial cells and fibroblasts^105,107. The first published clinical trial of Senolytic therapy (dasatinib plus quercetin, DQ) included 14 patients with IPF to evaluate feasibility of implementing a senolytic intervention (ClinicalTrials.gov ID: NCT02874989). DQ therapy had significantly improved the physical functions of the patients, (including 6-min walk distance, 4-m gait speed and chair-stands time). Based on these preliminary data, researchers are planning to carry out larger clinical trials of DQ therapy.

Growth factor applications

We could also induce the distal airway SSCs stemness by supplementing the essential stem cell growth factors and cytokines, whose availabilities are greatly reduced in chronic lung diseases. Exogeneous FGF7 (also known as keratinocyte growth factor) application could promote compensatory lung growth by alveolar proliferation post PNX in mice¹⁰⁸. A randomized controlled phase-I clinic trial showed that intravenous application of FGF7 attenuated alveolar injury in patients with acute respiratory disease syndrome (ARDS)¹⁰⁹. However, FGF7 application did not efficiently ameliorate any of the disease manifestations of ARDS in a phase-II clinical trial¹¹⁰.

Human populations with haplo-insufficiencies for FGF10 have higher incidence to develop COPD,¹¹¹ and FGF10 expression is suppressed in mesenchymal stromal cells from patients with IPF¹⁰³. Rodent studies have proven the therapeutic values of FGF10 in pulmonary epithelial regeneration in various lung disease models, such as bleomycin-induced alveolar injuries¹¹², naphthalene injury,¹¹³ and high-altitude pulmonary edema¹¹⁴. Well-designed clinical trials are needed to demonstrate the curative effects of FGF10 on distal airway regeneration in patients with chronic lung diseases.

Therapeutics Interventions on SSCs Microenvironment

As progress of tissue fibrosis limits the supportive functions of mesenchymal stromal cells to SSCs in distal airways, antifibrotic agents could be instrumental to distal airway epithelium repair. Quercetin induces apoptosis of senescent fibroblasts in aged mice with pulmonary fibrosis¹⁰⁷. Another study used a combination of quercetin and dasatinib (a tyrosine kinase inhibitor, widely known for its role in the treatment of myeloproliferative syndrome) to treat age-related morbidity in mice. The quercetin-dasatinib therapy significantly suppressed lung tissue fibrosis and aging of alveolar epithelial cells in aged mice¹⁰⁵. Navitoclax, a specific inhibitor of anti-apoptotic BCL-2 and BCL-XL, can selectively kill senescent cells in vitro. Navitoclax treatment effectively eliminated radiation-induced senescent fibroblasts and inhibited senescence-associated secretory phenotype (SASP) in mice¹¹⁵.

Growth factors, such as HGF and FGF1/2/7/10, can inhibit lung tissue fibrosis by reducing collagen accumulation in mice with pulmonary fibrosis^{112,116–119}.

All-trans retinoid acid (ATRA) is known as a key regulator of both embryonic lung development and postnatal alveolarization, promoting alveolar septum and lung growth¹²⁰. In vitro, ATRA stimulates human pulmonary angiogenesis and induces lung fibroblast elastin synthesis¹²¹. Preclinical studies using in vivo animal models of emphysema have demonstrated the incredible ability of ATRA to improve the state of emphysema and repair the alveolar structure of emphysema mice¹²². There was another trial showing that ATRA treatment of elastase-induced emphysema (Box 1) mice increased the number of reparative bone marrow-derived cells in the alveoli¹²³. Moreover, a recent study also supports some novel retinoid X receptor (RXRs) agonists as potential therapeutic strategies for the treatment of porcine pancreatic elastase and cigarette smoke extract (CSE)-induced emphysema¹²⁴.

Stem Cell Therapies

Stem cell transplantation has been proposed as an alternative cell therapy for IPF and COPD^125–127. Preclinical studies have shown that induced pluripotent stem cells (iPSCs), human embryonic stem cells (hESCs), mesenchymal stem cells (MSCs), or lung stem cells (LSCs) could be used to treat COPD and IPF^127–129. Transplantation of AT2s into bleomycin-induced IPF mice can reduce collagen deposition, restore pulmonary surfactant protein production, and alleviate the severity of pulmonary fibrosis¹²⁵. Similarly, lung injury was abrogated in bleomycin-induced IPF mice after transplantation of hESCs-derived AT2 cells¹²⁸. IPSCs transplantation into IPF mice could significantly improve the pulmonary functions¹³⁰. Furthermore, IPSCs transplantation bypasses possible ethical concerns and has a broader application prospect in comparison with ESCs. In a cigarette-smoke-stimulated COPD model, intrapulmonary injection of MSCs can alleviate airway inflammation and emphysema by down-regulating COX-2/PGE2 in alveolar macrophages¹³¹.

Challenges and Perspectives

The human respiratory system is anatomically different in details from that of mouse. The human lung has two left lobes and three right lobes, while the mouse lung has one left lobe and four right lobes. In mice, the conducting airway directly transits to the alveoli at the junction of the bronchial alveolar duct, while in humans and other mammals such as ferrets, the proximal airway gradually transitions to the farthest alveolar space through RB and alveolar tubules⁵. The pseudostratified columnar airway epithelium is present in the trachea and mainstem bronchus in mice, while it even extends to the distal bronchioles in human¹³².

The composition and proportion of SSCs within the distal airway epithelium varies between humans and mice. Most of the epithelial in the trachea are ciliated cells in human, but are nonciliated cells in mice¹³³. There are more goblet cells and fewer club cells in human airways than in mice. The newly discovered RAS cells within human-specific RB are absent in mice⁵. The distal BADJ region in the mouse lung contains BASCs population, whose presence has not been determined in human lung³². Since most of our studies and knowledge on lung homeostasis and regeneration are derived from mouse lungs, the differences between human and mouse lung tissues have limited our understanding of molecular and cellular heterogeneity in human lungs and hindered the investigations of pathogenesis of human chronic lung diseases and the development of related targeted therapeutics. We need to find and use other animal models, such as ferrets, whose lung structures and SSCs are closely related to human lungs⁵.

We still know little about the RB, which are particularly vulnerable to damages that are associated with chronic lung diseases. Distal airways are the target region of early injuries during COPD pathogenesis, and RB are the main obstruction sites of COPD^91,134, reminding us of the importance to unveil the pathogenesis of distal airways during early disease stages.

Footnotes

Acknowledgements

The authors would like to express their gratitude to Professor Pixin Ran from Guangzhou National Laboratory and Professor Jiekai Chen from Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences for the constructive comments to the manuscript.

Author Contributions

JW and HX conceived and wrote the manuscript. GP drew the graphics.

Ethical Approval

This study was approved by our Institutional Review Board.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article, and informed consent is not applicable.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Natural Science Foundation of China (grant no. 32070918), Distinguished Young Scholars in Guangdong Province (grant no. 2022B1515020109), Start-up funding for the Pediatric Research Institute of Guangzhou Women and Children’s Medical Center (grant no. 3001082).

ORCID iDs

Huahua Xu

Guihong Pan

Jun Wang

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