Single-Cell Transcriptomics Reveals Stem Cell-Derived Exosomes Attenuate Inflammatory Gene Expression in Pulmonary Oxygen Toxicity

Comments 1: [I would suggest replacing ”pulmonary oxygen poisoning” with ”pulmonary oxygen toxicity” throughout the paper, as this is the more used term].

Response 1:  Thank you for pointing this out. I still want to follow the academic language habits of our department for these years. Oxygen toxicity is a disciplinary disease, and my research is a branch of it.

Comments 2: Page 2, Lines 61-62: could you please expand on these exosomes?

Response 2:  I agree with this comment. I have made some supplementary instruction in the discussion section on page 12, Line 382-398. And you can also find some expand on exosomes on Page 2, Lines 65-74.

Comments 3: Page 2, Line 89 – Page 3, Line 93: how were these conditions/values chosen? Was this protocol following an established protocol?

Response 3: The experiment was designed with three groups to systematically evaluate the effects of hyperbaric oxygen (HBO) exposure on lung injury and the potential protective role of exosomes. The groups were as follows:

l Control group (CTRL): Mice receiving no HBO exposure and no exosome treatment, serving as the baseline.

l Hyperbaric oxygen group (HBO): Mice subjected to hyperbaric oxygen exposure without any therapeutic intervention, to assess the direct impact of HBO on pulmonary tissue.

l HBO + Exosome group (HBO+Exo): Mice pretreated with human mesenchymal stem cell-derived exosomes prior to HBO exposure, to investigate whether exosomes can mitigate or prevent HBO-induced lung injury.

This design allowed for the evaluation of two main variables: (1) the pathological effects of HBO on lung tissue, and (2) the potential prophylactic efficacy of exosomes in preventing or reducing such damage. Pairwise comparisons between the groups (CTRL vs. HBO, HBO vs. HBO+Exo, and CTRL vs. HBO+Exo) were conducted to determine the individual and interactive effects of HBO exposure and exosome administration.

Comments 4: Why were human exosomes used and not mouse?

Can you discuss the significance of using human exosomes in mice? Do you think the results would be different when using mouse exosomes?

Response 4:

Broader Clinical Translational Potential of hMSC-Derived Exosomes

Preclinical Relevance: The use of human-derived exosomes more accurately reflects potential therapeutic applications in humans, thereby enhancing the translational value of preclinical animal studies.

Alignment with Future Applications: Since this study aims to explore therapeutic strategies intended for human use, employing hMSC-derived exosomes in animal models provides more clinically relevant insights.

2   Functional Conservation of Human-Derived Exosomes

Cross-Species Conservation: Numerous studies have demonstrated that many functional components of exosomes, such as miRNAs and proteins, are highly conserved across species. Therefore, human-derived exosomes can exert biological functions effectively in murine models.

Supporting Literature: A growing body of published research has successfully utilized hMSC-derived exosomes in murine disease models with positive therapeutic outcomes (relevant references can be cited here).

3   Technical and Practical Advantages

Ease of Acquisition and Processing: hMSCs derived from sources such as umbilical cord, bone marrow, or adipose tissue are easier to culture and expand in vitro, and the process of exosome isolation from these cells is relatively well-established and reproducible.

Better Quality Control: Exosome products derived from hMSCs are subject to more standardized quality control procedures in both academic and industrial settings, facilitating reproducibility and component characterization.

4    Limitations of Murine MSC-Derived Exosomes

Isolation and Stability Challenges: Murine MSCs are difficult to isolate, have limited expansion potential, and are prone to senescence and differentiation, making it challenging to obtain sufficient quantities of high-quality exosomes.

Lack of Standardization: There is currently no consensus on the standard protocols for the purification, characterization, or quality control of murine-derived exosomes, which may compromise experimental consistency and reproducibility.

5    Additional Statement

Although interspecies differences exist, previous studies have shown that hMSC-derived exosomes retain robust bioactivity and demonstrate a favorable safety profile in murine models, supporting their use as a viable therapeutic agent in preclinical disease intervention studies.

Comments 5: Please expand more on the significance of the histological analysis of kidney cells and the results regarding the kidney cells.

Response 5: Thank you for pointing this out. Sorry for it’s a clerical error, I have convert lung instead of kidney in all the article.

Comments 6:Some of the results from Figure 1 are not described at all in the Methods section… IL-1β and TNF-α by ELASA, for example.

Response 6:  I have induce a paragraph in the methods section to describe IL-1β and TNF-α by ELASA on page 13 and 14 Line 136-150.

4. Response to Comments on the Quality of English Language

Point 1:

Response 1:   I have submit my manuscript in the MDPI website to ask for help on language editing.

5. Additional clarifications

[Hope to receive your response as soon as possible.]

1. Line 17: ‘The aim of this study was to investigate the protective mechanism of stem cell exosomes against lung

tissue and the potential molecular regulatory network.’

Line 20: ‘In this study, we employed single-cell RNA sequencing techniques to elucidate the unique cellular and

molecular mechanisms underlying the progression of exosome therapy for pulmonary type oxygen poisoning.’

There seems to be a syntax errors in the sentences above. Not sure what they are trying to

convey.

Also, please be consistent with use of ‘pulmonary oxygen toxicity’.

Yes. We have thoroughly reviewed the relevant sentences to ensure terminological consistency and corrected any grammatical inaccuracies (line17 and line21). Thank you for your meticulous review of our work.

2. Line 23: MSC-sEV not defined.

Line 24: POT not defined.

Please define acronyms at first sight of use, including the abstract.

Line 28: ‘In addition, our study of intercellular communication showed that there was significantly less

intercellular signal transduction between the POT group compared to the exosome treated group.’

What is intercellular communication?! This is a generic terminology not specifically defined previously and seems unrelated to other results presented in the abstract.

Thank you for your review of our manuscript and your valuable feedback. In accordance with your suggestions, we have provided the full terms and definitions for "MSC-sEV" and "POT" upon their first occurrence in the text (lines 24-25), and we have made corresponding modifications to the term "intercellular communication" (line 30) to ensure clarity and its correlation with other results.

3. Lines 53 – 58:

Not sure what is the difference between – when exposed to excess oxygen (line 53) and when

exposed to slightly elevated oxygen levels for long period of time (line 57)?

The description of acute and chronic oxygen poisoning is unclear and needs to be reframed

Thank you for your thorough review and feedback on our paper. We have made revisions to the relevant content to ensure that the descriptions are clearer (line 53-56).

4. Line 50 and 58: POT defined twice – should instead be first described in the abstract.

Yes, we have made revisions based on your suggestion and defined the term "POT" for the first time in the abstract to avoid repetitive definitions throughout the text.

5. Lines 61 – 63: ‘Current treatments inadequately address the molecular complexity of POT pathogenesis.

Exosomes, nanovesicles derived from mesenchymal stem cells (MSC-sEV), exhibit anti-inflammatory and

regenerative properties in lung diseases[9]. However, their role in POT remains uncharacterized.’

What current treatments and what molecular complexities?

The flow from current treatments, molecular complexities to introduction into exosomes here is

unclear and ill-defined. Please reframe

Thank you for the valuable feedback, Reviewer. We have revised the relevant paragraphs according to your suggestions to present a clearer explanation of the specific methods and limitations of current treatments for POT, as well as the complexity of the pathological mechanisms involved. This has allowed us to introduce the potential role of MSC-sEV (line 51-71).

6. Line 64: MSC acronym was already used in text so far but being defined first time here. Please correct.

Thank you for your feedback! We have revised the abbreviation "MSC" in the text to avoid the issue of repetitive definitions.

7. Line 65: What is low-frequency therapeutic modality? – please correct this.

Thank you for your meticulous review and feedback. Regarding the term "low-frequency therapeutic modality" mentioned in line 65, we recognize that this terminology was used inappropriately and have made the necessary revisions in the manuscript to adopt a more suitable expression. I sincerely apologize for any confusion this may have caused and appreciate your understanding and support.

8. Lines 65 – 69: The description of exosomes provided here is not adequate or appropriate given

its key context in this paper. Please expand pertinently.

Thank you for your feedback on our manuscript. In response to your concern regarding the insufficient description of exosomes, we have made the necessary revisions in the manuscript. In the latest version, we have expanded the definition and functions of exosomes and included their potential applications in various diseases.

9. Line 74: scRNA-seq has not been defined.

Thank you for your valuable feedback. We have supplemented the definition of scRNA-seq (single-cell RNA sequencing) in the revised manuscript.

10. Lines 75 – 79: ‘The purpose of this study was to investigate the effects of exosomes pretreated mice with

mesenchymal stem cells on hyperbaric oxygen-pulmonary oxygen poisoning by regulating pulmonary inflammation

and apoptosis, in an attempt to provide basic data and potential intervention strategies for the application of

exosomes in oxygen poisoning.’

There is a significant syntax error in the sentence above, rendering it difficult to interpret. Please

correct this.

Thank you for your feedback and suggestions regarding our research. In response to your comments about grammatical errors in the sentences, we have made corrections in the manuscript to enhance its readability and clarity.

11. The introduction is somewhat inadequate in providing pertinent background to this work.

Please note:

The introduction should briefly place the study in a broad context and highlight why it is

important. It should define the purpose of the work and its significance, including specific

hypotheses being tested. The current state of the research field should be reviewed carefully and

key publications cited. Please highlight controversial and diverging hypotheses when necessary.

Finally, briefly mention the main aim of the work and highlight the main conclusions. Keep the

introduction comprehensible to scientists working outside the topic of the paper.

Yes, we greatly appreciate your suggestions regarding the introduction section and have made corrections based on your pointed-out shortcomings. Thank you once again for reviewing our paper and providing valuable feedback.

12. What are experimental reagents (section 2.1)? Animals (a sub-section under this section) are not reagents!

Yes, We have made the necessary revisions and removed the section as suggested. Thank you for your valuable feedback.

13. Line 86 – 87: ‘Female C57BL /6 mouse, weighing about 18g (Shanghai Bikai Laboratory Animal Company), kept

in the Navy Medical University Animal Center.’

Please correct this sentence.

Thank you for reviewing our paper and providing feedback. We have revised the sentences to enhance their accuracy and academic quality.

14. Line 87 – 93: ‘The animals were divided into 3 groups: air control group, intravenous injection of phosphate

buffer saline (PBS) as control group, no hyperbaric oxygen exposure; The hyperbaric oxygen exposure group was

injected with the same amount of PBS as the control group, and exposed to 0.20 MPa, ≥95% oxygen for 8 h; In the

hyperbaric oxygen exosome intervention group, 100 μg exosomes were injected intravenously 1 day in advance, and

then treated with the same hyperbaric oxygen exposure group.’

The description of control and experimental groups is not very clear. There is a grammatical

error here.

What was the n for each group?
Why was intravenous injection of PBS given to the control group and hyperbaric oxygen exposure group?

Why was only 100 μg dose of intravenous exosomes injection studied in the experimental group?

What about other doses?

Why was this experimental group injected only 1 day in advance?

Thank you for your thorough review of our manuscript and your valuable comments.

1) This study is divided into three groups, utilizing a total of 18 mice, with a sample size of n=6 in each group.

2)The Control group received an injection of phosphate-buffered saline (PBS) as a negative control to eliminate any potential effects of the solvent (PBS) on the results. Similarly, the Hyperbaric Oxygen Exposure group was also injected with the same volume of PBS to ensure that the only experimental variable was the hyperbaric oxygen exposure, thereby excluding any interference from the injection procedure itself.

3) The choice of a 100 μg dose of exosomes in this study is supported by relevant literature, where this dosage has been shown to effectively demonstrate the biological effects of exosomes in previous research.

4) According to studies on the pharmacokinetics of exosomes^[1], 24 hours post-intravenous injection is the time at which exosomes reach peak concentration in lung tissue. Therefore, we chose to administer the exosomes one day prior to hyperbaric oxygen exposure to ensure that the exosomes could exert their effects effectively during the treatment process.

In response to your concerns regarding the clarity of the descriptions for the control and experimental groups, as well as the grammatical errors, we have made revisions to the manuscript (line 114-125).

1. Wang J, Yeung BZ, Cui M, Peer CJ, Lu Z, Figg WD, et al. Exosome is a mechanism of intercellular drug transfer: Application of quantitative pharmacology. J Control Release 2017; 268:147-158.

15. Line 104: ‘Western blot was performed to confirm the ex- pression of exosomal markers CD63 and CD9.’ And

calnexin (line130)

The results section only describes 1 western blot in figure 1C for TSG101, CD9 and CD81, that

do not correlate to the description in the methods section.

Also, no controls shown or described.

How does western blot for expression of exosomal markers CD63 and CD9 relate to and suffice

characterization of human umblical cord mesenchymal stem cell derived exosome?

Thank you for your feedback and suggestions. We have updated the Methods section to describe the Western blot as follows: "Detection of exosomal markers TSG101, CD9, CD81, and the negative control Calnexin." In Figure 1C, we indeed included the detection of Calnexin (as a loading control) to confirm the absence of organelle contamination (such as residual endoplasmic reticulum) in the exosomal samples. We have also revised the Results section to clearly state: "Calnexin was not detected in the exosomal samples, ruling out contamination from cellular debris."

According to the guidelines issued by the International Society for Extracellular Vesicles (ISEV) (MISEV2018), the characterization of exosomes requires at least three transmembrane or cytosolic markers (such as CD9, CD63, CD81, TSG101) and one negative marker (such as Calnexin or GM130). This study assessed CD9, CD81 (transmembrane proteins), and TSG101 (ESCRT complex), thereby meeting the minimum standards set forth by the guidelines.

16. Line 108: ‘Formalin-fixed, paraffin-embedded, 2 μm thick kidney sections were stained with H&E (Masson and

Sirius Red), and their histological score was determined.’

What was the purpose of performing the histological analysis of kidney sections and not lung

sections?

No related data is provided in the results section.

Thank you for your thorough review of our study and your valuable feedback. We acknowledge the errors in the Methods section and have made the necessary corrections to the manuscript.

17. Line 143: ‘Single cells were washed with PBS, and the cell number were analyzed using (Countess Auto Counter)

and viability (Trypan).’

Please correct syntax error here.

Thank you for pointing out the grammatical error. We have revised the sentence accordingly.

18. Line 153: Title is not appropriate. It implies only tSNE was used for dimensionality reduction,

while in description PCA is also utilized.

Thank you for your review and feedback on our manuscript. We have made revisions in the manuscript regarding the issue you raised about the title being inaccurate.

19. Line 157: ‘Genes were placed into 20 bins based on their average expression and removed using 0.0125 low

cutoff and 0.3 high cut-off.

Line 159: A z-score cutoff of 0.5 was applied to identify the highly variable genes, resulting in a total of 1,140

genes.’

Please describe why were these specific cut-offs used?

Thank you for your review and valuable comments on our manuscript. In response to your inquiry about gene selection methods and threshold choices, we provide the following detailed explanations:

1) Seurat defaults to categorizing genes into 20 bins to correct for the dependency between mean expression and variance-to-mean ratio. This approach effectively avoids inflated variance for highly expressed genes caused by technical noise (such as sequencing depth bias), thereby allowing for a more equitable identification of biologically relevant variable genes.

2) Regarding the choice of thresholds, a low cutoff of 0.0125 is used to exclude low-expressed genes that fall within the lowest 1.25% of the mean expression range. Such genes are prone to interference from technical noise due to their low signal-to-noise ratios, so we chose to exclude them to enhance the accuracy of the analysis. The high cutoff of 0.3 is employed to exclude highly expressed genes in the top 30% of the mean expression range. These genes are often housekeeping genes (e.g., Actb, Gapdh) that, despite having high expression levels, exhibit low variability and may obscure signals from biologically functional highly variable genes.

3) Within each expression bin, we calculate the z-score of gene dispersion (standardized variance/mean ratio) and filter for genes with z-scores greater than 0.5. This threshold balances sensitivity and specificity, ensuring the identification of genes with dispersion significantly higher than the background distribution at the same expression level (p < 0.05, assuming normal distribution). We found that this threshold effectively captures functionally relevant genes significantly associated with pathological phenotypes, such as inflammatory factors and repair-related genes. We believe that these choices enhance the accuracy and reliability of gene selection and provide important biological information for subsequent analyses.

20. Line 160, 161: Define PCA and PC.

Thank you for your valuable comments; we have made revisions in the manuscript accordingly.

21. Line 164: ‘tSNE visualized the single cells on a two-dimensional space based on expression signatures of the

variable genes, and therefore similar to POT loadings.’

What does this mean?!

Thank you for your feedback! We carefully reviewed the manuscript and found that there was indeed a typographical error in the description, which has now been corrected (line 227). We appreciate you pointing out this issue!

22. Overall, please provide a description of the single cell sequencing pipelines/workflow, including

library prep, barcoding. Include details of the reagents and technology used.

Followed by this the data control and dimensionality reduction description can be provided.

Thank you for your suggestions! I have added a detailed description of the single-cell sequencing workflow to the manuscript, including library preparation, barcoding technology, and the reagents and related techniques used.

23. Line 173 – 176: ‘Since different cells in the kidney share some well-known markers (transitional cells vs.

intercalated cells and proximal tubule vs. novel cells), the combination of these two approaches using the lower

threshold enabled us to retain the shared markers while identifying distinct markers compared to other cells.’

&

Lines 186 – 192: Then DBSCAN was used to identify cell types on the tSNE map with an initial eps value of 0.5.

Briefly, though 6 steps (①Preparation of single renal cell suspension, ②Single cell RNA sequencing: library

construction and quality control, ③Data quality control and preprocessing, ④Dimensionality reduction and tSNE

visualization, ⑤Identification of differentially expressed genes and marker genes, ⑥Cell clustering analysis:

dimensionality reduction analysis and tSNE map showed that renal cells were divided into 12 clustering) annotated

the 12 populations in Fig. 2A.

These sections are confusing and misleading.

Does this work relate to the single cell sequencing and transcriptomics in the lung (described in

section 2.6-2.8) or was all the previous methods description, relating to transcriptomics,

associated with the renal tissue?

Why was the initial eps value of 0.5 chosen?

What dataset does - renal cells were divided into 12 clustering) annotated the 12 populations in

Fig. 2A - refer to? The figure 2A in results describes lung histiocytes.

Additionally, figure 2 is described here in text before figure 1?!

Thank you for your careful review and feedback on our manuscript.

1) This study primarily focuses on single-cell sequencing and transcriptomic analysis of lung tissue. The methods and results described in sections 2.6-2.8 are indeed related to lung tissue, not renal tissue. We will clarify this in the manuscript to avoid any confusion.

2) The choice of an initial eps value of 0.5 is based on our preliminary analysis of data distribution and references from the literature. In the DBSCAN algorithm, the selection of the eps value significantly influences the clustering results. Our analysis revealed that an eps value of 0.5 effectively identifies cell types while avoiding over-clustering. The subsequent adjustment of the eps value to 1 was made to optimize the clustering results, ensuring that we could capture larger cell populations.

3) The statement in the previous paragraph indicating that "renal cells were divided into 12 clusters" is incorrect; our study only involves cells from lung tissue. The cell populations shown in Figure 2A are all derived from the lungs. We will revise the manuscript to correct this misunderstanding and ensure that the descriptions are consistent with the dataset.

4) We sincerely apologize for any confusion caused. We have corrected the relevant errors in the manuscript to ensure content consistency.

Thank you once again for your valuable suggestions.

24. The methods section does not describe all experimental details as required by the journal –

Materials and methods should be described with sufficient detail to allow others to replicate and

build on published results. New methods and protocols should be described in detail while well

established methods can be briefly described and appropriately cited. Give the name and version

of any software used and make clear whether computer code used is available. Include any pre

registration codes.

Thank you for your review and feedback on our manuscript. We have made revisions in response to your comments, which we believe will enhance the quality of the manuscript.

25. Section 3.1:

Cryo-TEm was performed, and results are somewhat described here.  However, the details on

how these results were generated should be provided in materials and methods section with

enough detail to allow replication of methods by reader.

Thank you for your review and feedback on our manuscript. We have added a detailed description of the "Cryo-TEM" results in the Materials and Methods section as you suggested.

26. Line 208: ‘The MSC-sEVs under investigation expressed the proteins CD9, CD63 and CD81,

but were negative for calnexin and cytochrome C.’

Figure 1C only shows western blots for CD 81, CD9 and TSG101. The description of results provided here are not corroborated with the figure.

Thank you for your meticulous review and feedback on our manuscript. In response to your comment regarding the description of MSC-sEVs protein expression, we have revised the manuscript to ensure that the results are consistent with the Western blot data presented in Figure 1C.

27. Lines 209 – 212: ‘To clarify the protective effect of exosome treatment against hyperbaric lung injury, we

injected MSC-sEV into a rat model of pulmonary oxygen toxicity (POT). HE staining was performed on the lung

tissues of mice. HE staining was performed on the lung tissues of mice.

In the hyperbaric oxygen-exposed group, the destruction of alveolar structure was obvious, and the alveolar wall

was thickened with obvious inflammatory cell infiltration. In the hyperbaric oxygen exosome intervention group, alveolar wall thickening and inflammatory cell infiltration were less severe than that in the hyperbaric oxygen

exposed group (Figure 1E-H), suggesting that exosomes could reduce the inflammatory injury of the lungs.’

Methods section only describes performing histological analysis of renal tissue.

The description in text suggests data in figure 1 E-H relate to histological analysis, however,

figure 1 associated text describes these panels are levels of IL-1β, TNF-α detected through

ELISA? (described as ELASA) and mRNA levels of CXCL 10.

No descriptive is provided for panel H in figure text and no descriptive for panel D in text.

No statistical assessment is provided in the figures.

Thank you for your feedback on our manuscript. Based on your suggestions, we have made revisions to the manuscript. The Methods section now accurately describes the histological analysis of lung tissues, and we have corrected the figure text to indicate that panels D-E show the levels of IL-1β and TNF-α detected by ELISA, while panels F-G display the mRNA levels of CXCL10. We have also added a description for panel H. Statistical assessments have been included in the manuscript.

28. It is unclear how ‘MSC-sEV Attenuates Hyperoxic Lung Injury’ (section title) is corroborated

through the results in these panels - which describes only exosome morphology, partial western

blots for marker expression and then some inflammatory marker expression.

Thank you for your valuable feedback on our manuscript. We have revised the title based on your suggestions to more accurately reflect the content of the study.

29. Also, figure 1, panels D-H use acronyms POT, MSC-Exos + POT that have not been defined

previously in methods section. This should be included in section 2.1.1 – in description of

control and experimental groups.

Thank you for your thorough review and valuable suggestions regarding our manuscript. We have defined the abbreviations "POT" and "MSC-Exos + POT" in the Methods section to ensure clarity in understanding these terms. We appreciate your feedback once again.

30. Line 228 – 230: ‘The dataset of lung histiocytes was annotated using signatures of known

lineage markers (Fig. 2A), and each marker contained renal cells from cell clusters and lineages

in the control, POT, and MSC-sEV + POT groups(Fig. 2B and C).’

‘Annotated using signatures of known lineage markers’ – where was this described in methods

section? Or provide a reference.

‘Each marker contained renal cells (?) from cell clusters and lineages in the control?!

Given these issues, it is thus unclear how to interpret data in Figure 2 A-C.

Thank you for your valuable feedback on our manuscript. In response to your concerns, we have made several revisions. We added a detailed explanation of the specific methods used for annotating the lung tissue dataset with lineage markers in the Methods section (line 222). This clarification enhances the transparency of our annotation process and the rationale behind our choice of markers. Additionally, we acknowledge that the term "renal cells" was a descriptive error and have corrected it, ensuring that the appropriate terminology is used throughout the text to avoid any confusion for readers. We appreciate your suggestions, which will significantly improve the clarity and accuracy of our research.

31. Line 236 – 237: ‘The proportion of inflammatory cells decreased in the exosome treatment

group, which was confirmed by HE staining.’

Where is this graphical data and associated statistics corroborating this in figure 2? The H&E

stained slides are only qualitative.

Thank you for your thorough review and feedback on our manuscript. We acknowledge that there was an error in the original wording, and we have made corrections to clarify that the results of the HE staining represent only qualitative observations. We greatly appreciate your insights, which have helped us enhance the accuracy and quality of our paper.

32. Lines 237-242: ‘The NicheNet tool12 was then utilised to infer cellular communication specific to hyperbaric

oxygen-associated gene expression patterns, based on the expression of ligands, receptors, relevant pathway

components, and genomic targets of these pathways (Fig. 2E). The cellular communication inferences derived from

the differentially expressed genes in the hyperbaric samples suggest that the effects of hyperbaric oxygen are

primarily mediated by inflammatory signalling.’

Figure 2 does not contain panel E.

These results – ‘Interaction between the cell in POT fibrotic niche after MSC-sEV

administration’ - are not appropriately verified through the data described and provided in figure

2.

Thank you for your thorough review and feedback on our manuscript. After careful examination, we realized that there were indeed errors in the descriptions within the manuscript. Consequently, we have removed the relevant content to ensure the accuracy and consistency of the paper. Once again, we appreciate your valuable suggestions; your feedback has played a significant role in improving our manuscript.

33. Line 252: ‘We identified five different epithelial cell populations, including four bronchial epithelial cells-AT1

cells, AT2 cells, Club cells, and Ciliated cells (Figure 3A).’

Results in figure 3A and text describe 4 populations only

Yes. After careful review, we confirm that there are indeed only four epithelial cell populations. Accordingly, we have made the necessary revisions to the manuscript to ensure the accuracy of the information.

34. Line 253-254: ‘AT1-like cell populations highly express Pdpn, Cav1, Hopx, AqP5, Vegfa, Col4a3, Col4a4

(Figure3B-C).’

Figure 3, panels B and C do not show results for Cav1, Hopx, AqP5, Vegfa, Col4a3, Col4a4

expression in the AT1 populations.

Thank you for your feedback. After review, we confirm that there was an error in the descriptions of the cell population expressions in the manuscript. This discrepancy arose from issues in the correspondence between the results section and the figures. We have re-labeled the relevant content to ensure consistency between the text and the figures.

35. Line 254: ‘AT1 cells are down-regulated in the pathology group. After treatment with stem cell-derived exosomes,

AT1 cells were increased.’

No relating graphical data with statistics is provided for comparison across groups.

Thank you for your valuable suggestions. After reviewing the manuscript, we confirmed that there were indeed errors in the descriptions. Consequently, we have removed the relevant content to eliminate any potential misunderstandings. We sincerely apologize for any confusion this may have caused you.

36. Lines 256-260: ‘Heterogeneity could also be identified based on the marker genes of each cluster (Figure 3D).

Compared with the control group, the apoptotic signaling pathway in alveolar type 1 (AT1) cells was significantly

upregulated following hyperbaric oxygen-induced lung injury. After treatment with stem cell-derived exosomes, the

apoptotic pathway in AT1 cells was downregulated(Figure3E-F).’

It is unclear how the results described in this section are related to figure 3, panels D-F. No

comparison is provided across groups. The KEGG data across figures 3-6 does not have clarity

Thank you for your meticulous review of the manuscript and for your valuable suggestions. Regarding the issue you raised, we acknowledge that the description of Figure 3 in the results section was not sufficiently clear. Specifically, Figure 3E presents the KEGG enrichment analysis comparing the control group with the hyperbaric oxygen-induced lung injury group (POT), while Figure 3F illustrates the KEGG enrichment analysis between the POT group and the group treated with stem cell-derived exosomes (MSC-Exos). To address this issue, we have revised the relevant content to ensure that the descriptions in the results section are clearer and more comprehensible.

37. Lines 280-284: ‘KEGG enrichment analysis revealed that pulmonary oxygen toxicity induced the down

regulation of oxidative pathways in venous vascular endothelial cells. After treatment with stem cell exosomes, some

pathways related to reactive oxygen species were down-regulated(Figure4E-F). The KEGG enrichment analysis

results indicated that stem cell-derived exosomes treatment might resist cell apoptosis and alleviate inflammatory

injury by alleviating the oxidative stress within cells.’

This is not interpretable across figure 4 E-F.

Thank you very much for your feedback! We have carefully reviewed the manuscript and identified the errors in the descriptions, which have now been corrected accordingly.

38. Text in this section and panels A-C in figure 4 relate to the endothelial cells, while section title

also mentions expression of inflammatory genes, which is not corroborated.

Thank you for your review and suggestions! We have made revisions addressing the issues you mentioned to ensure consistency between the discussion of endothelial cells and the expression of inflammatory genes.

39. Line 296: Figure. 45-C – Is this a typo?

Figure 5 does not have panel labels.

Yes, we have made corrections in the text regarding the first description.

40. Lines 297 – 301: ‘The number of cells in the Col13a1 + Fibroblasts and Col14a1 + Fibroblasts subpopulations

increased during exosome treatment, but Pericytes1 was unaffected, whereas Pericytes2 decreased significantly

after treatment. As illustrated in Figures 4A-C, two distinct cell subpopulations were identified in fibroblasts.’

This data is not provided in figure 5.

Thank you for your feedback. In response to your concerns, we have removed the inaccurate descriptions and made the corresponding modifications.

41. Line 311 -312: ‘Based on known cellular markers, we identified six lymphocyte populations, including B cells, T

cells, NK cells, and ILC2 (Fig. 6A-C).’

How does Panel C relate to above result?

Thank you for your feedback. Panel C displays the markers for four main cell types: Cd79a, Cd4, Cd8a, and Cxcr6, which validate our identification of B cells, T cells, NK cells, and ILC2 cells.

42. Lines 312 – 321:

‘After treatment with stem cell-derived exosomes, the number of NK cells, B cells, CD4+ T-cells, and CD8+ T-cells

decreased.

No statistics are provided relating to the description of these results in the graph.

Thank you for your thorough review of our work and your valuable feedback. Upon examining the manuscript, we recognized that the expression was not sufficiently accurate; therefore, we have revised the relevant content to ensure the accuracy and clarity of the data.

43. Differential expression gene analysis showed that the largest gene expression changes occurred in B cells and

CD8+T cell groups, and the altered pathways were involved in processes such as inflammatory response, cell cycle,

and oxidative phosphorylation (Fig6D- F).

Largest gene expression changes occurred in B cells - How was this result interpreted from the

data shown in figure 6?

Thank you for your feedback. In response to your comment regarding the inaccurate description of gene expression changes in B cells, we have made the necessary revisions to the manuscript.

44. Altered pathways were involved in processes such as inflammatory response, cell cycle, and

oxidative phosphorylation – which figure panel relates to this result?

NK cells, CD8+T cells, and Treg cells were predicted to be active lymphocytes that send signals to interact with

other cell types in a hyperoxic environment (Fig2e).

Description of NK cells, CD8+T cells, and Treg cells is not associated with Figure 2E.

Thank you for your feedback. We have made detailed revisions to the results section of the manuscript to ensure the accuracy and consistency of the information.

45. The results suggest that hyperoxygen alters adaptive immunity in the lungs by activating the innate immune system,

specifically causing B cells and CD4+The decrease in the number of T cells makes myeloid cells the main group of

immune cells in the lungs.’

Not sure what this means. This sentence is unclear

Thank you for your feedback. In response to your concerns about unclear sentences, we have revised the descriptions.

46. Section title is – ‘Stem cell exosome treatment inhibits lymphoid NK, CD8+ and CD4+ T cells in

mice’, however results described in text states that largest gene expression changes occurred in B

cells and CD8+T cell groups.

Thank you for reviewing our paper and for your suggestions. We have revised the title to more accurately reflect the research findings.

46. Section title is – ‘Stem cell exosome treatment inhibits lymphoid NK, CD8+ and CD4+ T cells in

mice’, however results described in text states that largest gene expression changes occurred in B

cells and CD8+T cell groups.

Thank you for your feedback. We recognize the inconsistency between the title and the results description in the manuscript. To address this issue, we have revised the title to: “Stem Cell Exosome Treatment Inhibits Lymphoid NK, B Cells, CD8+, and CD4+ T Cells in Mice.”

47. Overall, multiple interpretations drawn by authors are not sufficiently and precisely described in

the results and figures provided.

Additionally, figure quality is inadequate/low, rendering reading legends and labels inside the

panels very difficult.

Thank you for your feedback on our manuscript. We have reevaluated the results section to ensure that the descriptions of multiple interpretations are more accurate and clearer, and we have added further details. Regarding the quality of the figures, we have redone them to enhance clarity and readability, ensuring that the legends and labels are clearly visible. We greatly appreciate your constructive suggestions, which will help us improve the quality of the manuscript.

48. Lines 330 – 336: These are better fit for introduction than discussion.

The results described in previous sections are inadequately discussed here, rendering the

interpretation of these results incomplete. Authors should discuss the results and how they can be interpreted in perspective of previous

studies and of the working hypotheses. The findings and their implications should be discussed

in the broadest context possible. This section may be combined with Results.

Thank you for your review of our paper and your valuable feedback. In response to your comment regarding the insufficient discussion in the results section, we have reorganized the discussion, integrating the results with the discussion and adding comparative literature to clarify our findings and their significance more clearly.