Many tissues in the body are capable of regenerating by replacing defective or worn-out cells with new ones. This process relies heavily on stem cells, which are precursor cells that lack a set role in the body and can develop into different types of cells under the right conditions. Tissues often have their own pool of stem cells that they use to replenish damaged cells. But as we age, this regeneration process becomes less effective.

Many of our organs, such as the lungs, are lined with epithelial cells. These cells form a protective barrier, controlling what substances get in and out of the tissue. Alveoli are parts of the lungs that allow oxygen and carbon dioxide to move between the blood and the air in the lungs. And alveoli rely on an effective epithelial cell lining to work properly.

To replenish these epithelial cells, alveoli have pockets, in which a type of epithelial cell, known as AEC2, lives. These cells can serve as stem cells, developing into a different type of cell under the right conditions. To work properly, AEC2 cells require close interactions with another type of cell called L-MSC, which supports the maintenance of other cells and also has the ability to differentiate into several other cell types. Both cell types can be found close together in these stem cell pockets. So far, it has been unclear how aging affects how these cells work together to replenish the epithelial lining of the alveoli.

To investigate, Chanda et al. probed AEC2s and L-MSCs in the alveoli of young and old mice. The researchers collected both cell types from young (2-3 months) and aged (22-24 months) mice. Various combinations of these cells were grown to form 3D structures, mimicking how the cells grow in the lungs.

Young L-MSCs formed normal 3D structures with both young and aged AEC2 cells. But aged L-MSCs developed abnormal, loose structures with AEC2 cells (both young and old cells). Aged L-MSCs were found to have higher levels of an enzyme (called Nox4) that produces oxidants and other ‘pro-aging’ factors, compared to young L-MSCs. However, reducing Nox4 levels in aged L-MSCs allowed these cells to form normal 3D structures with young AEC2 cells, but not aged AEC2 cells.

These findings highlight the varying effects specific stem cells have, and how their behaviour is affected by pro-aging factors. Moreover, the pro-aging enzyme Nox4 shows potential as a therapeutic target – downregulating its activity may reverse critical effects of aging in cells.

Substantial progress has been made in our understanding of the biology of aging, and these advances have the potential to improve both healthspan and lifespan, while alleviating the burden of age-related diseases. Self-organization in biological systems is a process by which cells reduce their internal entropy and maintain order within these dynamic, self-renewing systems (Chatterjee et al., 2017; Kiss et al., 2009). Exhaustion of stem/progenitor cells, cellular senescence, and altered intercellular communication have been proposed as aging hallmarks that increase susceptibility to age-related disorders (Schultz and Sinclair, 2016). However, the interactions between these hallmarks and whether cellular bioenergetics associated with cellular senescence may account for age-associated stem cell dysfunction and altered cell-cell communication have not been well defined. In this study, we utilized an organoid model to study stem cell behavior and intercellular communication that may account for age-related phenotypes; through these studies, we identify cellular bioenergetics and redox imbalance as critical drivers of these inter-dependent aging hallmarks.

The mammalian lung serves an essential role in organismal metabolism, uniquely by serving as the primary organ for systemic exchange of oxygen for carbon dioxide. Regeneration and maintenance of structure-function of the lung are dependent on adult, tissue-resident stem cells that reside in unique niches along the airways (Basil et al., 2020; Hogan et al., 2014). Type 2 alveolar epithelial cells (AEC2s) serve as facultative stem/progenitor cells in adult mammalian lungs and differentiate into type 1 alveolar epithelial cells (AEC1s) in response to diverse injuries to reconstitute and reestablish the alveolar gas exchange surface (Barkauskas et al., 2013). AEC2 regenerative capacity declines with age, resulting in impaired lung injury repair responses, thus increasing susceptibility to various lung diseases (Schulte et al., 2019; Watson et al., 2020). To further explore the relationship between AECs and lung mesenchymal stromal cells (L-MSCs), we developed an alveolosphere assay system that has been traditionally used to assess AEC2 stemness or regenerative potential (Barkauskas et al., 2013). In this assay system, L-MSCs and AEC2s are mixed together in a ratio of 100,000–5000,, respectively, and seeded in Matrigel (Figure 1A); alveolospheres with a single layer of epithelial cells composed of both AEC2s (surfactant protein C [SFTPC]-positive) and AEC1s (lung type I integral membrane glycoprotein [T1α]-positive) surrounding a hollow sphere typically form 9–12 days following co-culture (Figure 1B and C). L-MSCs expressing platelet-derived growth factor receptor-α (PDFGRα), with a fewer number expressing α-smooth muscle actin (α-SMA), were found primarily in cells lining the outer edges of alveolospheres (Figure 1D and E). AEC2s within alveolospheres stained for both the cell proliferation marker, Ki-67, and the double-strand DNA damage repair marker, histone H2A.X, suggesting ongoing AEC2 turnover within these 3D organoids (Figure 1F and G). This self-organizing behavior of L-MSCs and AEC2s is critically dependent on the presence of both cell types as AEC2s in the absence of AEC2s do not self-organize to form alveolospheres (Figure 1—figure supplement 1A). Together, these data support the crosstalk between L-MSCs and AEC2s that permit formation of distinct alveolar organoid-like structures; this intercellular communication may be perturbed during aging accounting for age-associated loss of AEC2 regeneration/maintenance.

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Alveolosphere formation with varying combinations of L-MSCs and AEC2s.

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To determine the effect of age on cellular self-organization and alveolosphere formation, L-MSCs and AEC2s were isolated from the lungs of young (2–3 months) and aged (22–24 months) mice and seeded in various combinations (Figure 1H, upper panel). L-MSCs and AEC2s from young mice generated normal alveolospheres, whereas L-MSCs and AEC2s from the lungs of aged mice showed impaired self-organization with condensed, poorly formed organoids. Interestingly, combining aged AEC2s with young L-MSCs resulted in relatively normal-appearing alveolospheres, while the reverse combination (young AEC2s and old L-MSCs) did not (Figure 1H, middle and lower panels; Figure 1—figure supplement 1B). Quantitative analyses revealed that, while the number of alveolospheres formed was not significantly different between groups (Figure 1I), the size of alveolospheres was critically dependent on the age of L-MSCs, and not that of AEC2s (Figure 1J). These findings indicate that secreted factor(s) from young L-MSCs are capable of supporting the self-organizing behavior of alveolospheres, while aged L-MSCs do not.

Cellular senescence accumulates in tissues with advancing age (Krishnamurthy et al., 2004) and has been proposed as a key driver of aging and aging-related disease phenotypes (Baker et al., 2016; Kennedy et al., 2014). Based on our observation that aged L-MSCs were incapable of supporting alveolosphere formation, we explored whether the emergence of cellular senescence may account for this finding. First, we confirmed the senescent features of L-MSCs from aged mice in monolayer 2D cell culture (Figure 2A, Figure 2—figure supplement 1) and in 3D alveolospheres (Figure 2B) by staining for β-galactosidase (β-gal) (Dimri et al., 1995) and lipofuscin (Georgakopoulou et al., 2013), respectively. To characterize secreted factors that may account for the age-associated dysregulation of cell-cell communication, we first measured cytokines/growth factors secreted from both young and aged L-MSCs using antibody arrays (Figure 2C). A number of cytokines that have been associated with the senescence-associated secretory phenotype (SASP) were found to be released at higher levels by aged L-MSCs, including IL-6, CCL12/MCP-5, CCL11/Eotaxin, and WISP-1/CCN4; in contrast, IGFBP1 was the only secreted protein that was statistically more elevated in young L-MSCs in this cytokine array (Figure 2D and E).

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Cytokine array and mass spectrometry data comparing young and aged L-MSCs.

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In addition to predefined cytokine array analyses, we employed an unbiased approach to identification of secreted proteins from young and aged L-MSCs by mass spectrometry-based discovery proteomics (Figure 2F). After adjustments for false discovery rates, 503 high-confidence proteins were identified; of these, 235 proteins were found to have non-zero quantifiable values in at least three of four experimental repeats per group for subsequent statistical analysis. The 36 proteins that passed both a single pairwise statistical test (p<0.05) in addition to a fold change of ±1.5 (Supplementary file 1A) were subjected to principal component analysis (PCA) (Figure 2G) and heat map analysis (Figure 2H; quantitation of heat map proteins, Figure 2I). Gene ontology localization analyses revealed enrichment in secreted proteins, including extracellular vesicles and extracellular matrix (Supplementary file 1B); interestingly, gene ontology processes analysis indicated the top cellular processes as negative regulation of reactive oxygen species metabolic process and extracellular matrix/structure organization (Supplementary file 1C). Enrichment by top toxic pathologies revealed proteins associated with lung fibrosis (Supplementary file 1D), a disease associated with aging (Thannickal et al., 2014). Network analysis revealed upregulation of pathways associated with the canonical WNT-signaling pathway and cell cycle regulation (Supplementary file 1E). Together, these findings suggest that aged L-MSCs are characterized by cellular senescence associated with SASP factors, oxidative stress, and alterations in regenerative pathways that may account for impaired alveolosphere formation.

Altered cellular metabolism has been linked to both senescence and aging (López-Otín et al., 2016; Finkel, 2015; Sun et al., 2016). To compare the energy state between young and aged lung L-MSCs, rates of mitochondrial respiration and glycolysis were analyzed by Seahorse XF Analyzer. Real-time oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were significantly higher in aged L-MSCs as compared to young L-MSCs (Figure 3A and B). Aged L-MSCs showed significantly higher basal, maximal, ATP-linked, proton leak, and non-mitochondrial respiration when compared to young L-MSCs with no change in reserve capacity (Figure 3C-H). Higher non-mitochondrial OCRs in aged L-MSCs suggest potential activation of oxygen-metabolizing NADPH oxidases in aged L-MSCs. An energy map profile of the OCR and ECAR data indicated a higher basal rate of both mitochondrial respiration and glycolysis (Figure 3I), suggesting that aged L-MSCs are under a basal state of higher metabolic demand. This higher energy demand under conditions of equivalent nutrient supply was associated with reduced levels of ATP/ADP under steady-state basal conditions (Figure 3J). Measurement of ECAR indicated that, despite similar basal ECAR, higher rates of maximal and non-glycolysis related ECAR were observed in aged L-MSCs. (Figure 3—figure supplement 1). Together, these data indicate that senescence of L-MSCs is characterized by higher baseline metabolic demand with reduced bioenergetic efficiency.

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Bioenergetics of senescent L-MSCs.

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Hydrogen peroxide (H₂O₂) has emerged as critical regulator of redox signaling and oxidative stress (Sies, 2017). Replication-induced senescence of human lung fibroblasts results in higher rates of extracellular H₂O₂ release in association with increased expression of NADPH oxidase 4 (Nox4) (Sanders et al., 2015), a gene that is inducible by the pro-senescent/pro-fibrotic mediator, transforming growth factor-β1 (TGF-β1) (Hecker et al., 2009). We explored whether L-MSCs isolated from naturally aged mice are associated with higher Nox4-dependent H₂O₂ secretion; in comparison to young (3 months old) L-MSCs, aged (22–24 months old), L-MSCs released higher baseline levels of H₂O₂, an effect that was further stimulated by exogenous stimulation with TGF-β1 (Figure 4A). This age-dependent, pro-oxidant phenotype of L-MSCs was markedly reduced in aged mice heterozygous for Nox4 (Nox4^-/+; Figure 4B and C), implicating Nox4 as a critical mediator of both basal and TGF-β1-induced H₂O₂ release. Although it is difficult to replicate the tonic, continuous release of H₂O₂ from a constitutive enzymatic source such as Nox4 in metabolically active cells, we tested the effect of a bolus addition of H₂O₂ (1 µM) to organoid culture comprising young AEC2s and young L-MSCs; this resulted in a decrease in the size of alveolospheres, without significantly affecting the numbers of alveolospheres formed (Figure 4—figure supplement 1A–C).

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Functional characteristics of wild type and Nox4-deficient aged L-MSCs.

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Recent studies implicate Nox4 in metabolic reprogramming (Bernard et al., 2017), although the effects of Nox4 on age-related metabolic dysfunction are unclear. The reduced levels of ATP/ADP in aged L-MSCs were partially rescued in age-matched Nox4^-/+ L-MSCs (Figure 4D), implicating a role for Nox4 in the reduced bioenergetic efficiency associated with aging. The steady-state levels of the tricarboxylic acid (TCA) cycle metabolites, malate and citrate, were reduced in wild-type aged L-MSCs; these levels recovered to levels similar to young L-MSCs in Nox4^-/+ L-MSCs, although only malate achieved statistical significance (Figure 4E and F). Levels of other TCA metabolites that were not significantly altered with aging in L-MSCs were not affected by a deficiency in Nox4 (Figure 4—figure supplement 1D-G). β-gal activity was found to be significantly lower in Nox4-deficient aged L-MSCs as compared to wild-type aged L-MSCs (Figure 4—figure supplement 1H). In addition to metabolic dysfunction, an important feature of senescence reprogramming is the activation of a SASP program. We determined whether Nox4 modulates the release of cytokines/growth factors by aged L-MSCs. Protein (antibody-based) arrays showed significant downregulation of SASP-associated cytokines in aged Nox4^-/+ L-MSCs vs. aged L-MSCs (Figure 4—figure supplement 1I). We were unable to identify a group of cytokines (except for coagulation factor III) that were specifically identified in the dataset comparing young vs. aged L-MSCs based on the antibody-based cytokine array method. Further studies with an unbiased approach such as that employed by mass spectrometric analyses (Figure 2I) will be required for more in-depth analyses of Nox4 regulated SASP cytokines/growth factors. Interestingly, Nox4 haploinsufficiency in aged L-MSCs resulted in significant downregulation of OCR as compared to aged L-MSCs (Figure 4G). Nox4^-/+ aged L-MSCs showed significantly lower basal, ATP-linked, proton leak, and maximal respiration compared to wild-type aged L-MSCs. Non-mitochondrial respiration was also lower in aged Nox4^-/+ L-MSCs, although this did not achieve statistical significance (Figure 4H). Together, these studies indicate that Nox4 contributes to metabolic dysfunction, bioenergetic inefficiency, and SASP programming in L-MSC aging.

Based on the observations that Nox4 alters cellular bioenergetics of L-MSCs, we examined the effects of Nox4 on regulating the self-organizing potential of alveolospheres. The aberrant formation of alveolospheres when young AEC2s were cultured with aged L-MSCs were rescued when replaced with age-matched Nox4^-/+ L-MSCs (Figure 4I; Figure 4—figure supplement 1J); quantitation of both alveolospheres counts (Figure 4J) and alveolosphere size (Figure 4K) was significantly enhanced when these assays were conducted with Nox4-deficient L-MSCs. We were also unable to detect significant changes in alveolosphere formation when the Nox4 inhibitor, GKT137831 (10 µM), was added to an organoid culture system of young AEC2s and aged L-MSCs (Figure 4—figure supplement 1K-M); however, it is difficult to ascertain whether this intervention effectively inhibited Nox4 activity in this organoid model. While the phenotype of impaired alveolosphere formation in young AECs and aged L-MSCs was reversed by haploinsufficiency of Nox4 in L-MSCs (Figure 4I–K), this was not sufficient to completely reverse the phenotype in aged AEC2s (Figure 4—figure supplement 1N-P). This finding, along with the observation that wild-type young L-MSCs are sufficient to reverse the aged AEC2 phenotype (Figure 1H), suggests that in addition to reduced expression of Nox4 in aged L-MSCs, the young mesenchyme may support niche function and recovery of alveolosphere formation of aged AEC2s through Nox4-independent mechanisms. Thus, an aging alveolar stem cell niche may involve both a gain of ‘pro-senescent factors’ (Nox4) and loss of ‘rejuvenation factors’ by the mesenchyme; the identification of the latter will require further study. Together, our studies support a critical role for Nox4-dependent oxidative stress, senescence, and bioenergetic insufficiency in restricting the capacity for cellular self-organization in a 3D organoid model of aging and stem/progenitor cell function (Figure 4L).

A fundamental difference between non-living matter and life forms on our planet is the ability to extract energy from the environment, primarily through oxidation of carbon-based fuels. Thus, the second law of thermodynamics that governs both living and non-living matter does not strictly apply to living organisms capable of self-renewal through the continuous and dynamic exchange of energy (from exogenous nutrients) and mass (biosynthetic and degradative processes in respiring tissues/organs). It follows then that, when this capacity for energy-dependent self-renewal is diminished, the law of entropic degeneration will be operative in living organisms. Aging is associated with derangements in metabolism that influences, and may in fact control, many of the well-recognized hallmarks of aging, including cellular senescence (López-Otín et al., 2016; López-Otín et al., 2013). The inter-relatedness of metabolism with these traditional aging hallmarks is difficult to untangle due to complexities of studies in living organisms and the relative simplicity of 2D cell culture models.

Organoids offer the opportunity to study complex living phenomena such as emergent properties and self-organization in biological systems while at the same time allowing for reductionist approaches that provide mechanistic understanding of these complex processes. Using a combination of methods that included a 3D organoid model, biochemical approaches, proteomics, and gene targeting, we demonstrate a critical role for the oxygen metabolizing enzyme, Nox4, in regulating cellular bioenergetics and senescence that limits stem cell function. Consistent with contemporary theories on aging, Nox4 may function as an antagonistically pleiotropic gene and contribute a number of age-related degenerative disorders, including fibrotic diseases (Thannickal, 2010; Lambeth, 2007). Interestingly, proteomic analyses of proteins secreted by aged L-MSCs in the current study suggested alterations in cellular redox/oxidative stress (from gene ontology analysis) and lung fibrosis (as a ‘toxic pathology’). These findings, as well as candidate SASP factors, highlight the emerging importance of intercellular communication and stem cell exhaustion as important hallmarks of aging (López-Otín et al., 2013).

Regenerative mechanisms in adult, mammalian organisms primarily rely on the activation and differentiation of tissue/organ-resident stem cells (Hogan et al., 2014). Homeostatic maintenance of these stem cells requires a tightly regulated niche that includes other supporting cell types, in particular stromal cells (Basil et al., 2020). An important finding from our studies is the observation that L-MSC aging, relative to AEC aging, largely accounts for the age-related inability to form alveolospheres. Thus, aging of the stem cell niche may be as important, and perhaps more decisive, in promoting certain age-related pathologies. Therapeutic targeting of metabolic aging within the stem cell niche, specifically that of lung-resident fibroblasts/myofibroblasts, may offer a more effective and viable strategy for age-related degenerative disorders such as tissue/organ fibrosis.

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Author details

1. Diptiman Chanda

   Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

   For correspondence

   dchanda@uab.edu

   Competing interests

   none

   "This ORCID iD identifies the author of this article:" 0000-0002-4835-4460

2. Mohammad Rehan

   John W. Deming Department of Medicine, Tulane University School of Medicine, New Orleans, United States

   Contribution

   Investigation, Software, Writing – review and editing

   Competing interests

   none

3. Samuel R Smith

   Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States

   Contribution

   Data curation, Formal analysis, Software, Writing – review and editing

   Competing interests

   none

4. Kevin G Dsouza

   Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   none

5. Yong Wang

   Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States

   Contribution

   Investigation

   Competing interests

   none

6. Karen Bernard

   Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States

   Contribution

   Methodology, Writing – review and editing

   Competing interests

   none

7. Deepali Kurundkar

   Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States

   Contribution

   Methodology, Resources

   Competing interests

   none

8. Vinayak Memula

   1. Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States
   2. Department of Surgery, Birmingham, United States

   Contribution

   Formal analysis, Software

   Competing interests

   none

9. Kyoko Kojima

   Comprehensive Cancer Center Mass Spectrometry & Proteomics Shared Facility, Birmingham, United States

   Contribution

   Formal analysis, Investigation, Methodology, Resources

   Competing interests

   none

10. James A Mobley

    Department of Anesthesiology and Perioperative Medicine, Birmingham, United States

    Contribution

    Formal analysis, Methodology, Resources, Software, Writing – review and editing

    Competing interests

    none

11. Gloria A Benavides

    Department of Pathology, Birmingham, United States

    Contribution

    Formal analysis, Methodology, Resources, Software

    Competing interests

    None

12. Victor Darley-Usmar

    Department of Pathology, Birmingham, United States

    Contribution

    Data curation, Formal analysis, Resources, Supervision, Writing – review and editing

    Competing interests

    none

13. Young-iL Kim

    1. Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States
    2. Division of Preventive Medicine, Department of Medicine; University of Alabama at Birmingham, Birmingham, United States

    Contribution

    Formal analysis, Resources, Writing – review and editing

    Competing interests

    none

14. Jaroslaw W Zmijewski

    Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States

    Contribution

    Writing – review and editing

    Competing interests

    none

15. Jessy S Deshane

    Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States

    Contribution

    Resources, Writing – review and editing

    Competing interests

    none

16. Stijn De Langhe

    Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Birmingham, United States

    Contribution

    Writing – review and editing

    Competing interests

    none

    "This ORCID iD identifies the author of this article:" 0000-0003-3867-4572

17. Victor J Thannickal

    John W. Deming Department of Medicine, Tulane University School of Medicine, New Orleans, United States

    Contribution

    Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review and editing

    For correspondence

    vthannickal@tulane.edu

    Competing interests

    none

    "This ORCID iD identifies the author of this article:" 0000-0003-4266-8677

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