Avalilação PREreview de Lung progenitors’ exhaustion in response to microenvironment stress during aging

Review coordinated via ASAPbio’s crowd preprint review

This review reflects comments and contributions by Joseph Biggane, Diptarup Mallick, Teena Bajaj, Randa Salah Gomaa Mahmoud, Danielle Rayee, Annajiat Alim Rsael, Sophie Richard, Yuka Gliday, Yohana Amos, Maria Vidal, Reuben Opoku, Nitya Khetarpal, Priya Dutta, Shailee Rasania, Chinyere Mary-Cynthia Ikele, Emmanuel Odame, and Hala Taha Elbashir. Review synthesized by Joseph Biggane.

This study investigates how aging affects lung progenitor cell regeneration by examining epithelial–fibroblast interactions using lung organoid models from young, aged, and accelerated-aging (Zmpste24-/-) mice, demonstrating that epithelial progenitors retain regenerative capacity while senescent fibroblast niches, altered autophagy, and reduced nuclear lamins contribute to impaired regeneration and increased susceptibility to mechanical stress. This study presents several interesting and novel findings, which, taken together, represent a significant contribution to the regeneration and aging fields.

Crowd Review Comments:

• Pertaining to the Abstract:

  • In response to “...we used aged wild-type mice of 18-24 months old, and Zmpste24-/- deficient mice…”, the authors should consider identifying the abbreviation ZMPSTE24 as a zinc metalloprotease.

  • In response to “...particularly a subpopulation of alveolar epithelial type 2 (AT2) that can restore alveolar structure…”, it may be helpful to briefly define why AT2 cells are particularly relevant to regenerative studies (e.g., their role in surfactant production and alveolar repair).

• Pertaining to the Introduction:

  • In response to “Despite recent advances in understanding adult lung stem cells…”, the reviewers suggest that this paragraph outlining the methodological approach might be moved to the end of the Introduction, after the background has been presented, and immediately before the study aim. This would improve the overall flow of the section.

  • In response to “...Zmpste24-deficient mice (a model of premature aging)...”, what is the rationale for this mouse model over other models of premature aging? Why choose a whole-mouse "reduction" rather than a lung-specific KO?

  • In response to “Autophagy maintains cellular homeostasis by removing damage or unnecessary cytoplasmic components through the lysosomal pathway.”, it might be of interest to indicate how autophagy preserves the stemness of AT2 cells, specifically.

• Pertaining to the Results:

  • In subsection ‘Progenitor epithelial cells of aged mice hold the regenerative potential’...

    • In reference to “...selected with the EpCam marker by magnetic beads (MACS)”, the MACS acronym is not clearly introduced. The authors should include the entire abbreviated name of the technology.

    • In reference to “...the majority displayed a type II phenotype...”, the readers assume this refers to alveolar type II cells, but the nomenclature is inconsistent with the introduction (these were declared as AT2 cells there). Further, many of the proteins have been introduced in the results without the context of utility. This makes the article unnecessarily unapproachable to a broad audience.

    • In reference to “...surfactant protein C (Sftpc)...”, in the methods, surfactant protein C is referred to as SPC.

    • In reference to Figure 1, the statement that "aged cells retain regenerative potential" is potentially overstated. Organoid formation alone does not fully capture functional regeneration (e.g., differentiation efficiency, long-term self-renewal).

    • In response to “...epithelial cells derived from young wild-type mice (12–16 weeks), physiologically aged wild-type mice (18–24 months), and Zmpste24-deficient mice, which exhibit accelerated aging (16 weeks) and with 10,000 pre-cultured fibroblasts”, a lot of the subsequent data shows similarities between the WT aged mice and the Zmpste24-deficient mice. The authors mention that the Zmpste24-deficient mice are displaying signs of aging, like graying, but should it be expected that the results, like autophagy, be the same as those of advanced-aged mice?

    • In response to “...isolated epithelial cells from both aging models are capable of forming organoids…”, lung progenitor cells are known to express specific transcription factors such as Sox9 (Rawlins, 2008). The authors don't show specific markers in the organoid cultures.

    • In response to “...sizes comparable to those generated by young wild-type mice when co-cultured with fibroblasts from young wild-type mice”, organoid size is not actually demonstrated or quantified in Figure 1. Size quantification could not be located in other main figures or the supplemental figures.

    • In response to “...organoid growth curve was performed with old and young wild-type cells…”, the authors might consider a change in language to something more precise, like "...growth curve measuring the number of organoids...".

  • In subsection ‘Autophagy is upregulated in epithelial cells from aging-accelerated mice’...

    • The reviewers suggest that the authors consider that increased autophagy gene expression is not equal to increased functional autophagy flux. There was no protein-level validation (e.g., LC3-II, p62) in epithelial cells.

    • In response to “A volcano plot further revealed that Zmpste24-/- mice cells had an increased expression of autophagy genes such as LC3, Atg12, Atg3, and Lamp1. Notably, AKt1, a negative regulator of autophagy, is found to be significantly downregulated in Zmpste24 deficient mice…”, it would be valuable to include and further discuss the volcano plot of autophagy-related genes in naturally aged mice, in addition to the Zmpste24 model. This would help clarify whether the observed pattern is conserved beyond the heatmap representation in both aged models.

  • In subsection ‘Senescent niche limits lung organoid growth’...

    • This section refers to a niche, but only fibroblasts are evaluated. Is it possible to isolate clones, or more clearly define how these differ? This is a strong section of the paper!

    • In response to “we evaluated senescence by SA-βgal activity, which showed a significant increase of senescent cells in Zmpste24-/- fibroblasts…”, have the authors also considered evaluating SASP markers?

    • In response to “...autophagy flux analyzed through LC3 and p62 after chloroquine (CQ) treatment…”, the time course of this experiment is unclear. The panel indicates +/- CQ for 24h, but flux implies more variability than one time point.

    • In response to Figure 3, the reviewers could not identify an explanation for what is seen in the aged WT fibroblasts.

    • In response to Figure 4, the reviewers suggest that the authors maintain the WT control throughout since the zmpste24 mutant/KO is a case of accelerated aging.

    • In response to Figure 5A, it is clear that nuclear Lamin A/C is reduced in the WT old; however, this isn't the case in the genetically driven accelerated aged mice Zmpste. Also, the number of nuclei analyzed should be specified.

  • In subsection ‘Mechanical stress from FACS impairs epithelial cell regenerative potential’...

    • In response to “...difference between these studies and ours is that while previous work relied on FACS sorting to isolate epithelial cells, we used only magnetic bead-based isolation…”, a stronger potential comparison would be to show the integrity of the cells after MAC alone, MAC+FACS, FACS alone. This could be via morphology in immunofluorescence, more epithelial markers (not just γH2AX, but a flow cytometry panel, for instance), or immunostaining.

• Pertaining to the Discusssion:

  • In response to “Taken together, these findings support the notion that aging may trigger a hormetic response in the lung, characterized by resistance to senescence and activation of autophagy.”, it seems as if the authors are proposing that the increase in autophagy is a mechanism to protect against aging in the stem populations. This seems counterintuitive to the reviewers because the whole idea of the mitochondrial theory of aging is that there is a decrease of autophagy in aged stem cells. If this is the explanation being proposed, then the authors should include references here to point to other examples of this phenomenon.

  • In response to “Although this link between lamin loss and progenitor cell status has not been previously studied in the lung, our findings provide initial evidence supporting a connection.”, the claim that the link between lamin loss and progenitor cell status is not particularly well-supported by the preceding paragraph relating to FACS sorting.

  • In response to “...microenvironmental signals coordinate an autophagy-driven stress response to promote regeneration.”, this claim does not have complete experimental justification. The authors haven't characterized how the organoids and fibroblasts are communicating with each other to put forward a claim that microenvironmental signals coordinate autophagy-driven stress response to promote regeneration.

• Pertaining to the Methods:

  • Biological sex of the mice should be included along with any potential biasing of the data (i.e., differences between females and males).

  • In subsection ‘RT-qPCR’, the authors should consider including thermocycling parameters for reproducibility purposes.

  • In subsection ‘Immunofluorescence’, the authors should include each antibody concentration used for reproducibility purposes.

• Pertaining to the Supplementary Figures:

  • In subsection ‘Supplemental Figure 1a’, the reviewers suggest replacing "growth" with "number". "Growth" here is misleading because it might induce readers to think that the authors quantified the size of the organoids.

  • In subsection ‘Supplementary Figure 3b’, it would be worthwhile to provide some sort of details about the number of fibroblasts, use a double axis, or graph separately. The fibroblast numbers are simply too small to see any differences between the groups.

  • In subsection ‘Supplementary Figure 6a’, it would be beneficial to label which mouse is which.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.