Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
(1) As mentioned above, numerous studies have reported that the number of MuSCs declines with aging. The authors' claim is valid, as Pax7 and Vcam1 were widely used for these observations. However, age-related differences have also been reported even when using these markers (Porpiglia et al., Cell Stem Cell 2022; Liu et al., Cell Rep 2013). (a) When comparing geriatric Vcam1⁺ MuSCs with young MuSCs in this study, did the authors observe any of the previously reported differences? (b) Furthermore, would increasing the sample size in Figure 1 reveal a statistically significant difference? The lack of significance appears to result from variation within the young group. (c) In addition, this reviewer requests the presentation of data on MuSC frequency in geriatric control mice using CD200 and CD63 in the final figure.
(a) When comparing geriatric Vcam1+ MuSCs with middle aged MuSCs, we found 1,428 DEGs, where 701 genes were downregulated and 727 genes were upregulated (Fig. S3E). Some of the pathways altered were similar to previously reported differences, such as alterations in the autophagy-lysosome related genes and PI3K-Akt Pathways. However, these alterations did not affect the functional integrity of geriatric Vcam1+ MuSCs (Fig. 3 A-F). On the other hand, greater alterations were observed in geriatric Vcam1- MuSCs, accompanied by functional impairment. We have added further elaborations in the manuscript to reflect the comment from the reviewer (pg. 17, lines 369-379).
(b) Thank you for this helpful comment. We understand the reviewer’s concern that the variability within the young group may contribute to the absence of statistical significance. We respectfully note that the variance observed in the young cohort could be biologically expected rather than technical noise. Multiple studies have shown that young adult MuSCs display great transcriptional and functional heterogeneity from undergoing post-natal myogenic maturation (e.g., Biressi et al., 2010; Tierney & Sacco, 2016; Motohashi & Asakura, 2014). This broader heterogeneity naturally increases variance in marker distribution within young samples. We would also like to clarify that our main conclusions are not solely based on differences in the overall proportion of YFP⁺ and Lin⁻ cells among age groups. Instead, we also rely on the functional and phenotypic heterogeneity that specifically emerges in geriatric MuSCs.
Although the young group shows greater biological variation, the mean values are relatively similar among the groups. Multiple independent datasets in our study including functional performance and molecular profiles consistently show that the total MuSC frequency does not markedly decline with aging. For these reasons, even if the sample size is increased, we do not expect a change in the overall interpretation of this result. We have revised the Results section to acknowledge the variability observed in the young group and to emphasize that total MuSC frequency is not central to the conclusions of this study (pg. 6, lines 129-134).
(c) MuSC frequency in geriatric control mice using CD200 and CD63 in the final figure are in the figure legend of Fig. 5F (pg. 39, line 825-828).
(2) Can the authors identify any unique characteristics of Pax7-VCAM-1 GERI-MuSCs using only the data generated in this study, without relying on public databases? For example, reduced expression of Vcam1 and Pax7. The results of such analyses should be presented.
In Fig S2C, using the bulk-RNA sequencing data generated in this study, we observe reduced expression of both Pax7 and Vcam1 in Pax7-VCAM-1 GERI-MuSCs population. To better highlight this finding, we have added text in the Results section that explicitly describes the reduced Pax7 expression and Vcam1 loss as distinguishing features of Pax7-VCAM-1 GERI-MuSCs in our dataset (pg. 9, lines 199-200).
(3) In the senolysis experiment, the authors state that GER1-MuSCs were depleted. However, no data are provided to support this conclusion. Quantitative cell count data would directly address this concern. In addition, the FACS profile corresponding to Figure 4D should be included.
In Figure 4D we quantified the frequency of VCAM1 Low YFP positive Lin negative MuSCs after senolysis treatment. This analysis shows a clear trend toward a decrease in the GERI subpopulation, although the difference did not reach conventional statistical significance in this experiment (t test p = 0.0596). We have therefore revised the text to describe this as a reduction trend rather than complete depletion, and we now explicitly report the p value in the results section (pg. 12, line 270-272). Furthermore, representative FACS profiles for Figure 4D is now included with the quantification (pg. 38, line 811-814).
(4) Figure S4: It remains unclear whether DHT enhances regenerative ability through restoration of the VCAM1 expression in GER1-MuSCs, as DHT also acts on non-MuSC populations. Analyses of the regenerative ability of Senolysis+DHT mice may help to clarify this issue.
We thank the reviewer for this important insight. We agree that DHT can act on non-stem cell populations in the muscle environment and therefore we cannot conclusively attribute the improved regenerative performance solely to restoration of VCAM1 expression in GERI-MuSCs. To address this concern, we have revised the discussion to explicitly state this limitation and to clarify that DHT may influence multiple cell types that contribute to muscle regeneration. We also indicate that combined senolysis plus DHT treatment would be an informative future approach, although additional animal experiments were not feasible within the scope of the current study (pg. 18, line 382-390).
(5) Why are there so many myonuclear transcripts detected in the single-cell RNA-seq data? Was this dataset actually generated using single-nucleus RNA-seq? This reviewer considers it inappropriate to directly compare scRNA-seq and snRNA-seq results.
Regarding the question of why many myonuclear transcripts were detected and whether this dataset was generated using single nucleus RNA sequencing, we confirm that the experiments were performed using single cell RNA sequencing. The presence of myonuclear transcripts likely reflects partial nuclear leakage or fragmentation during the enzymatic dissociation of aged muscle tissue. This is a known technical issue when preparing single cell suspensions from adult or geriatric skeletal muscle.
To avoid inappropriate interpretation, we identified the myonuclear transcript enriched cluster and excluded it from all downstream analyses that involve MuSC comparison. Therefore, our major conclusions do not rely on this cluster. We have revised the Results text to clearly state that the dataset was generated using single cell RNA sequencing and to explain how myonuclear transcript-positive cells were handled (pg. 8, lines 176-181).
Reviewer #2 (Public review):
In this study, Kim et al. explore the heterogeneity within the aged MuSC population using a mouse model that enables lineage tracing of MuSCs throughout life. The questions addressed in the manuscript are highly relevant to the fields of aging and stem cell biology, and the experimental approach overcomes limitations of earlier studies. However, some of the claims would benefit from additional data analysis, and the central claim of the identification of a "previously unrecognized subpopulation" of aged MuSCs should be evaluated in light of prior work that has also examined MuSC heterogeneity in aging.
Specific points:
(1) As a general comment that is transversal to multiple figures, several experiments should include a direct comparison to a young cohort. Previous studies have shown that the depletion of subpopulations with aging is observed early in the aging process, for example, the loss of Pax7-high MuSCs is observed already in 18‐month‐old mice (Li, 2019, doi: 10.15252/embj.2019102154). Using only mice at 12-14 months as the control group is therefore insufficient to claim that no changes occur with aging.
We thank the reviewer’s suggestion for comparing the aged mice to a young cohort and we acknowledge that previous studies have observed depletion of subpopulations is observed early in the aging process. However, this study is specifically designed to delineate the transition from middle aged to geriatric stages, rather than to characterize differences that are already well established in young versus geriatric comparisons. Previous studies have extensively documented the decline in MuSC function between young and aged animals, whereas the process and timing by which these changes emerge remain unclear. Our results show that major alterations in MuSC phenotype and identity are detected predominantly in the geriatric stage rather than at the middle aged stage. To avoid any misunderstanding, we have revised the text to clearly state that the primary objective of this work is to define the critical shift that occurs from middle aged to geriatric muscle stem cells (page 3-4, line 67-71).
(2) One of the central claims of the manuscript is a challenge to the notion that MuSCs number declines with age. However, the data analysis associated with the quantification of YFP+ cells needs to be expanded to support this conclusion. The authors present YFP+ cells only as a proportion of Lin-neg cells. Since FAP numbers are known to decrease with aging, a stable proportion of YFP+ cells would simply indicate that MuSCs decline at the same rate as FAPs. To more accurately assess changes in MuSC abundance, the authors should report absolute numbers of YFP+ cells normalized to tissue mass (cells/ mg of muscle).
We thank the reviewer for this helpful suggestion. We agree that a proportion based analysis alone does not fully exclude the possibility that MuSCs and FAPs decrease at similar rates during aging. At the time of isolation, muscle mass was not recorded, so we are unable to report YFP+ cell numbers normalized to tissue weight as requested. To partially address this limitation, we have now clarified our gating strategy in the methods and Figure 1 to explicitly indicate Sca1+ FAP exclusion (pg. 6, line 121-122, pg. 22, lines 460-463). These analyses do not support a major selective loss of MuSCs relative to other mesenchymal populations with aging.
(3) The authors emphasize that several studies use VCAM1 as a surface marker to identify MuSCs. However, many other groups rely on α7-integrin, and according to Figure 1D, the decline in ITGA7 expression within the YFP+ population is not significant. Therefore, the suggestion that MuSC numbers have been misquantified with aging would apply only to a subset of studies. If the authors can demonstrate that YFP+ cell numbers (normalized per milligram of tissue) remain unchanged in geriatric mice, the discussion should directly address the discrepancies with studies that quantify MuSCs using the Lin−/α7-integrin+ strategy.
We thank the reviewer for this important comment. We agree that VCAM1 is only one of several commonly used surface markers for MuSC identification and that many studies quantify MuSCs using the Lin negative and ITGA7 positive strategy. That is why in our study, in addition to VCAM1, we also examined ITGA7 expression within the YFP positive population. Although the mean ITGA7 level did not significantly decline, the variance among geriatric MuSCs was significantly increased based on the F test. This supports the idea that aging does not uniformly reduce marker expression but instead increases phenotypic instability, which could lead to under detection of a subset of MuSCs even when ITGA7 is used as the primary marker. We have added this interpretation to the Discussion (pg. 16, lines 346-355).
(4) The authors focus their attention on a population of VCAM-low/VCAM-neg subpopulation of MuSCs that is enriched in aging. However, the functional properties of this same population in middle-aged (or young) mice are not addressed. Thus, it remains unclear whether geriatric VCAM-low/VCAM-neg MuSCs lose regenerative potential or whether this subpopulation inherently possesses low regenerative capacity and simply expands during aging.
We thank the reviewer for this comment. In young and middle aged mice, the VCAM low or VCAM negative population is extremely small, nearly absent in most samples. The emergence and expansion of this population is therefore a feature that becomes detectable only at the geriatric stage. Given that these cells are not present in appreciable numbers earlier in life, the reduced regenerative performance observed in geriatric VCAM1low MuSCs likely reflects a phenotype that arises during aging rather than an inherent property of a pre-existing subpopulation. We have added this clarification to the Results section (pg. 7, lines 142-146).
(5) According to Figure 1F, the majority of MuSCs appear to fall within the category of VCAM-low or VCAM-neg (over 80% by visual estimate). It would be important to have an exact quantification of these data. As a result, the assays testing the proliferative and regenerative capacity of VCAM-low/negative cells are effectively assessing the performance of more than 80% of geriatric MuSCs, which unsurprisingly show reduced efficiency. Perhaps more interesting is the fact that a population of VCAM-high geriatric MuSCs retains full regenerative potential. However, the existence of MuSCs that preserve regenerative potential into old age has been reported in other studies (Garcia-Prat, 2020, doi: 10.1038/s41556-020-00593-7; Li, 2019, doi: 10.15252/embj.2019102154). At this point, the central question is whether the authors are describing the same aging-resistant subpopulations of MuSCs using a new marker (VCAM) or whether this study truly identifies a new subpopulation of MuSCs. The authors should directly compare the YFP+VCAM+ aged cells with other subpopulations that maintain regenerative potential in aging.
We thank the reviewer for this comment. First, in response to the request for precise quantification, we now provide the proportions of VCAM1-high and VCAM1-low/negative MuSCs in each age group in the figure legends for Fig.1F (pg. 34-35, lines 765-772). In geriatric mice, VCAM1 low/negative MuSCs represent approximately 44.6% ± 35.7%, whereas VCAM-high MuSCs represent 3.9% ± 1.8%. The substantial variability reflects mouse-to-mouse heterogeneity at very advanced ages.
Importantly, our conclusions do not rely solely on the observation that a large fraction of geriatric MuSCs exhibit reduced regenerative potential. Rather, the VCAM-low state represents a transcriptionally and functionally distinct subpopulation that emerges specifically in the geriatric stage, and exhibits molecular signatures not present in young or mid-aged MuSCs. We have expanded the Results and Discussion to clarify this point.
Regarding whether VCAM-high geriatric MuSCs correspond to previously reported “aging-resistant” MuSCs (e.g., Garcia-Prat 2020; Li 2019), we agree that there may be conceptual overlap, as both populations retain regenerative activity. However, those studies identified resilient MuSCs based on mitochondrial or Pax7-high properties, whereas our classification is based on surface VCAM1 intensity, and we currently lack direct evidence that these populations are equivalent. We have therefore added a statement acknowledging this possibility while clarifying that our work does not claim that VCAM1-high MuSCs represent a newly discovered resilient subset, but instead focuses on the emergence and characterization of the VCAM-low dysfunctional subpopulation (pg. 16, lines 346-355).
(6) In Figure 3F, it is unclear from the data presentation and figure legend whether the authors are considering the average of fiber sizes in each mouse as a replicate (with three data points per condition), or applied statistical analysis directly to all individual fiber measurements. The very low p-values with n=3 are surprising. It is important to account for the fact that observations from the same mouse are correlated (shared microenvironment, mouse-specific effects) and therefore cannot be considered independent.
We thank the reviewer for raising this important statistical point. We fully agree that individual myofibers from the same mouse are not independent biological replicates. In morphometric analyses of regenerated muscle, however, it is standard practice to analyze the full CSA distribution across all regenerated fibers, as the distribution itself (rather than a per-mouse mean) provides the biologically relevant measure of regeneration quality.
The original analysis therefore treated each regenerated fiber as a component of the overall CSA distribution, not as an independent biological replicate, and the statistical comparison was performed at the level of distributions rather than per-mouse replication. We agree that per-mouse averaged CSA values would also be informative, but the raw data were not archived in a format that allows reconstruction of mouse-specific fiber subsets.
Importantly, the group-level CSA distribution differences are robust and remain clearly detectable regardless of statistical approach. We have added clarification in the figure legend to explicitly describe how CSA measurements were obtained and analyzed mouse (pg. 36, lines 796-800).
(7) Regarding Figure 5, it is unclear why ITGA7, a classical surface marker for MuSCs that appears unchanged in aged YFP+ MuSCs (Fig. 1F), is considered inadequate for detecting and isolating GERI-MuSCs.
We thank the reviewer for raising this point. As shown in Figure 1F, the mean ITGA7 expression level does not significantly decline in geriatric YFP positive MuSCs. However, the variance of ITGA7 expression is significantly increased in geriatric MuSCs based on the F test, indicating instability in surface marker expression. This suggests that a fraction of MuSCs may fall below the conventional gating threshold for ITGA7 during aging. Therefore, ITGA7 remains effective for identifying a large portion of MuSCs but may under detect the subset of geriatric MuSCs with reduced marker expression. We have revised the Discussion to clarify this point (pg. 16, lines 346-355).
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) Figure 3B: In the colony formation assay, the authors should specify the number of biological replicates and the number of cells analyzed per mouse.
We have now added the number of biological replicates and the number of cells analyzed per mouse in the figure legend of Figure 3B (pg. 37, lines 790-791).
(2) Figure 3F: The replication number is indicated as n = 3, which appears to refer to the number of transplanted mice. How many myofibers were analyzed in each transplanted mouse? The authors should provide a more detailed description of the methodology in the Figure legend or M&M.
We thank the reviewer for the question and clarify that n = 3 refers to three independent transplanted mice per group. For each mouse, the entire TA muscle was cryosectioned and immunostained, and all regenerated fibers containing centrally located nuclei were included in the CSA quantification. We have added clarification in the Figure legend to indicate that quantification was performed on all regenerated fibers from each mouse (pg. 37, lines 796-800).
(3) Figure 4: The RNA-seq results are presented as a single dataset per sample. If multiple experiments were performed, individual datasets should be shown. Replicated analyses are essential to ensure the reliability of the findings.
In response to the reviewer comment, we confirm that the RNA sequencing in Figure 4 was performed with 3-4 independent biological replicates for each condition. These replicates showed very consistent sequencing quality and gene expression profiles and were therefore combined for the differential expression analysis. We have revised the materials and methods to clearly describe the number of biological replicates and the analysis workflow. (pg. 25, lines 543).
(4) Line 148: If the authors examined MyoG expression, it should be described as committed myoblasts.
We have now changed the term from myoblasts to committed myoblasts (pg. 8, line 168).
(5) Typo and Referencing Errors:
(a) Line 244: The term 'Antide' appears to be a typo.
We thank the reviewer for noting this point. ‘Antide’ is not a typo but the correct name of a GnRH antagonist (Antide acetate). To avoid confusion, we have revised the text to specify ‘Antide, a GnRH antagonist’ at its first mention (pg. 13, line 289).
(b) Lines 278, 280: Please correct Figure 5H to Figure 5F.
We apologize for this error. We have fixed the figure notations accordingly (pg. 15, lines 326-330).
(c) Some references are incomplete or inappropriate (ex. line 49, line 71, line 86, line 109).
We apologize for this error. We have fixed the references accordingly (pg. 4, line 94, pg.6, line 117).
(d) Line 49: Skeletal muscle regeneration is orchestrated primarily by tissue resident stem cells, known as muscle stem cells (MuSCs) or satellite cells (Relaix et al., 2021). The following paper should be cited:
Satellite cell of skeletal muscle fibers.
MAURO A. J Biophys Biochem Cytol. 1961 Feb;9(2):493-5.
The reference has been revised (pg. 3, line 49).
(e) Line 109: Paired box protein 7 (Pax7) is a transcription factor widely recognized as a defining marker of MuSCs (Sambasivan et al., 2011). The following paper should be cited:
Pax7 is required for the specification of myogenic satellite cells.
Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. Cell. 2000 Sep 15;102(6):777-86.
The reference has been revised (pg.6, line 117).
(6) Lines 73-74: Many rejuvenation studies define 'aged' mice as 12 to 24 months old. This reviewer is not aware of any studies that have examined 12-month-old MuSCs as a model of aging.
We apologize for this error. We have fixed the numbers to 18 months accordingly (pg. 4, line 94).
Reviewer #3 (Recommendations for the authors):
(1) Geriatric versus aged mice in the MuSC subpopulation analysis. The authors use geriatric mice (>28 months) to demonstrate the loss of VCam expression in MuSCs and propose that this accounts for previous reports of decreased MuSC numbers in aged contexts. However, as noted in their introduction, most reports use "aged" mice, which are typically around 24 months old, which is biologically distinct from the geriatric stage. This distinction makes it difficult to conclude that the reported decline in MuSC numbers in aged mice can be explained by the phenomenon observed only in geriatric mice (Line 289). The authors should test whether VCam expression is altered in aged (24-month-old) mice to strengthen this argument.
We appreciate the reviewer’s thoughtful comment and agree that 24 month old mice are commonly used as an aged reference in the literature. However, prior studies using 18 to 24 month old animals have reported inconsistent results regarding whether and to what extent MuSCs decline during this period. To avoid ambiguity from intermediate aging stages, we purposefully selected geriatric mice older than 28 months, a condition under which MuSC depletion has been more consistently reported in previous studies. Notably, our data show that even at this stage MuSC abundance is not dramatically reduced, which makes it unlikely that a robust decline would already be present at 24 months. We have clarified this rationale in the revised text. Although investigating the precise timing of the emergence of these changes at earlier time points is an important future direction, it is beyond the scope of the present study.
(2) Variability and bimodal distributions.
Figure 1b: The decline in VCAM+ MuSCs in geriatric mice shows high variability - 3 of 7 replicates align more closely with young/mid-aged levels. Please clarify this variability.
We thank the reviewer for pointing out the variability. We agree that there is heterogeneity in the extent of VCAM1 reduction across geriatric mice. This variability likely reflects animal-to-animal differences in the onset and progression of aging-related phenotypes, which are known to vary at very advanced ages. Importantly, despite this variability, all geriatric samples contain a detectable VCAM1 low population that is not observed in young or middle-aged mice, and the overall trend is consistent across all replicates. We have clarified this in the revised manuscript (pg. 6, lines 125-127).
Figure 1c: While the Mid and Geriatric groups are tightly clustered, the Young group appears bimodal, which challenges the claim (Line 118) that values are "comparable across ages." Since all males were used and it is not sex related, what is driving this bimodal distribution?
We appreciate the reviewer’s observation regarding the variability in the young group. Muscle stem cells in young adult mice are known to encompass diverse transcriptional and functional substates, which contribute to greater biological heterogeneity at this stage (Biressi et al. 2010; Tierney & Sacco 2016; Motohashi & Asakura 2014). As aging progresses, these substates gradually converge toward a common functional phenotype, resulting in more uniform profiles in middle-aged and geriatric mice. Therefore the bimodal appearance in the young group likely reflects the broader developmental heterogeneity of early adult MuSCs rather than a technical discrepancy. We have added this explanation to the revised in the results section (pg.6. lines 129-134).
Figure 4D: Geriatric replicates also display a trimodal distribution. This should be addressed throughout - what is causing these types of distribution, and how does this impact significance tests and conclusions?
We appreciate the reviewer’s observation regarding the multimodal distribution. We interpret this pattern as reflecting increased individual variability that becomes more pronounced at the geriatric stage. Even though aging affects all mice, the extent and timing of age-related phenotypic changes can vary considerably across individuals at very advanced ages. This leads to broader divergence in VCAM1 expression states among geriatric mice. Therefore, when we look at the correlation between VCAM1 High and VCAM1 Low/- population, there exists a significant negative correlation between the two populations (Fig. S3F). We have clarified this interpretation in the text and note that the statistical analysis was performed using the mouse as the biological replicate, so this variability does not alter the overall conclusion (pg.12-13, lines 270-278).
(3) The fate of the Vcam-low/negative cells should be better assessed. For example, Line 180: Colony formation is low/absent in VCAM-low/- cells. Are these cells still viable? Cell death assays are needed. Is expansion capacity truly impaired, or are the cells simply non-viable? Using gene expression as the only means (Line 300) to suggest not dying is insufficient.
We thank the reviewer for this important point. As per the reviewer's analysis, there is lack of direct evidence to show that these cells are viable and apoptosis or viability assay would further strengthen our research. However, we carefully suggest that they are viable from the fact that these cells can be isolated by FACS and generate high quality RNA sequencing libraries, which would not be possible if they were undergoing cell death. Moreover, the transcriptomic data indicate upregulation of stress response and senescence associated pathways rather than apoptotic or necrotic signatures. These findings suggest that VCAM low or negative cells are alive but exhibit reduced proliferative and regenerative capacity. We have revised the text to clarify that our data reflect impaired function rather than loss of viability and that apoptosis assays represent a direction for future investigation (pg. 16, 360-366).
(4) Transplant assays are suggestive, but could use additional characterization. Lines 191 & Figure 3E-F: While representative images match quantification, areas at the edge of VCAM-low/- TAs show signs of regeneration. Please include lower-magnification images. Additionally, assess early post-transplant engraftment efficiency - do certain populations experience a higher loss rate (cell death)? YFP-tracing would also help confirm the donor contribution to fibers.
While we did not collect additional early time-point samples for new engraftment analyses, we carefully re-examined all available transplantation data, including the distribution and density of YFP+ donor-derived cells in early post-injury sections. We did not observe patterns suggestive of differential early cell loss between VCAM-high and VCAM-low groups. Thus, although we cannot formally quantify early engraftment efficiency, the existing evidence does not support a model in which differential donor-cell retention accounts for the observed regenerative differences.
Also, we attempted direct YFP co-staining of regenerated myofibers, but as reported by several groups, YFP signal within mature or regenerating myofibers is often diminished or inconsistent after fixation and permeabilization, making reliable fiber-level YFP detection technically challenging in our system. Therefore, instead, we confirmed donor contribution using PBS-injected control muscles, which lack donor MuSCs, and showed that PBS-injected muscles never generated YFP+ fibers. This demonstrates that endogenous MuSCs do not contribute to YFP⁺ myofibers in our model, and therefore indirectly supports our suggestion that any YFP⁺-regenerated fiber necessarily originates from transplanted donor cells. We hope the reviewer understands the technical limitations.
(5) Figure S3D: mRNA profiling suggests Mid-aged MuSCs are more distinct from Geriatric Vcam-hi than expected. This should be addressed or at least elaborated on in text.
We appreciate this insightful comment. We agree that mid aged VCAM high MuSCs show detectable transcriptional differences from geriatric VCAM high cells. This pattern likely reflects the fact that some aging related molecular changes begin to accumulate gradually during the middle aged stage even before overt functional decline or VCAM1 loss becomes evident. Importantly, however, these transcriptomic shifts do not lead to the emergence of the VCAM low dysfunctional phenotype that is uniquely present in geriatric muscle. We have added clarification to the text noting that molecular alterations arise progressively while the major phenotypic transition in VCAM1 expression and regenerative impairment occurs at the geriatric stage (pg.11, 238-244).
(6) The conclusion of senescence needs more support. Lines 218-226: p16 is elevated in VCAM-low/- cells, but drawing conclusions on senescence from 1-2 markers (mRNA) is insufficient. DQ Treatment: It's unclear how DQ alters cell composition in the absence of clear senescence markers (besides p16). Since DQ targets BCL-2/anti-apoptotic pathways, analyzing these signaling cascades is necessary. Line 255: The term "terminally senescent" is contradictory. These may be pre-senescent. It's also surprising DQ would target such cells, and further clarification is needed. Lines 307-313: Proposing a revised definition of senescence is premature. These cells may be pre-senescent, and multiple ways to senescence exist (replicative, stress-induced, etc.). Please clarify.
We agree with the reviewer that the term 'terminally senescent' may be premature and potentially contradictory. Although p16 is elevated in this population, we acknowledge that one or two mRNA markers are insufficient to establish bona fide senescence, and that multiple senescence programs exist, including replicative, stress-induced, and mitochondrial-associated pathways. We have revised this to 'senescent-like' throughout the manuscript to better reflect the complexity of this state. Also, although beyond the scope of this study, we now emphasize that future studies incorporating additional senescence markers, functional assays, and lineage tracing will be required to determine the precise senescence status of VCAM-low MuSCs (pg.17-18, lines 381-392).
Regarding DQ treatment, we agree that DQ is not selective for senescent cells, as it targets BCL-2–related survival pathways. The reduction of VCAM-low cells after DQ treatment therefore indicates increased dependence on survival signaling in this population rather than providing direct evidence of senescence. We have revised the text to clarify this interpretation (pg.12-13, lines 270-278).
(7) Figure 5C: The Pax7+ cells appear interstitial rather than sublaminar. This raises questions about the specificity of staining. Providing lower-magnification images with these as insets may help.
We thank the reviewer for this helpful comment. We agree that the high-magnification image in Figure 5C may give the impression that Pax7+ cells are interstitial due to the limited field of view. We regret to inform the reviewer that low-magnification images for this sample are not available as these images were obtained via confocal imaging where we only recorded areas of interest. Therefore, we are unable to provide an additional panel at this time and we hope the reviewer understand.
(8) CD63 and CD200 expression on Pax7-YFP traced cells. Figure 5: YFP-traced geriatric MuSCs co-stained for CD63 and CD200 are essential. Current data only show expression in Young traced cells. It's crucial to confirm whether protein/surface expression persists in geriatric YFP+ (traced) cells. The current Figure 5 F does not appear to include YFP tracing for geriatrics.
We thank the reviewer for highlighting the importance of confirming CD63 and CD200 expression specifically in Pax7-YFP traced MuSCs from geriatric muscle. The datasets shown in Figure 5F were generated from wild-type C57BL/6 mice using a standard MuSC gating strategy rather than Pax7-YFP animals. All geriatric Pax7-YFP mice available for this study were exhausted during earlier experiments, and additional tissue is not available for new co-staining or FACS analyses. We now state this technical limitation in the manuscript and clarify that the geriatric CD63/CD200 data were obtained from conventionally isolated MuSC populations rather than YFP-traced cells (pg.18-19, lines 407-416).
Minor points:
(1) Please show the outliers in addition to the concentric circles. Figures 1B, C, and F are examples, but this should be addressed throughout.
Outliers have been added where applicable.
(2) Figure 2C: Was a significance test performed between the 5 dpi and "geri" fractions?
We thank the reviewer for this important point. We have now performed the requested statistical comparison between the 5 dpi fraction and the geriatric VCAM1-defined subpopulations using the same analysis framework applied in Figure 2 (Kruskal–Wallis test followed by Dunn’s multiple comparisons).
While 5 dpi MuSCs differed significantly from young MuSCs (adjusted p = 0.0139), the comparisons between 5 dpi and each geriatric subgroup (VCAM-high, -mid, and -low) did not reach statistical significance after correction for multiple testing (adjusted p = 0.17, 0.15, and 0.17, respectively). These results have been added to the revised Figure 2C corresponding figure legend (pg. 36, lines 777-780).
Importantly, we now clarify in the text that although 5 dpi muscles display a prominent increase in VCAM1-high cells at the population level, this increase does not statistically exceed the variability observed within geriatric subpopulations under the conservative non-parametric testing framework used.
(3) Line 155: The phrase "Surprisingly, all clusters mapped to quiescent clusters" is misleading; this is expected given the population type.
We thank the reviewer for this helpful comment. We have revised the sentence to remove the misleading wording and now describe the observation more accurately (pg. 8 lines 180-181).
(4) Line 211: The figure notation should be corrected from Figure S4E to Figure S3E.
We apologize for this error. We have fixed the figure notation for Figure S4E to S3E (pg. 11, line 247).
(5) Line 216: "All of which" seems overstated. Many populations share similar profiles with minor differences.
We appreciate the reviewer’s comment. We agree that the phrase “all of which” overstated the degree of divergence among clusters. We have revised the wording to more accurately reflect the data (pg. 11-12, lines 252-253).
(6) Line 270: The notations for panels D, E, and F need to be updated to match the figure. Panel "H" is not indicated in Figure 5.
We apologize for this error. We have fixed the figure notations accordingly (pg. 15, lines 326-336).
