PONE-D-24-47284Comparative Analysis of Extracellular Vesicles from Induced and Adipose-Derived Mesenchymal Stem Cells: Implications for Regenerative Medicine

PLOS ONE

Dear Dr. Ababneh,

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Please address and incorporate the following queries and suggestions, along with the concerns raised by the reviewer. "The section under this heading “Generation of Embryoid Bodies from iMSCs (EB-iMSCs)” should be divided into two distinct parts to improve clarity and flow. The second part should focus on the protocol for generating embryoid bodies (EBs) from induced Mesenchymal Stem Cells (iMSCs), including a detailed step-by-step process for embryoid body formation. This would include key factors such as cell seeding densities, culture conditions, and the duration of the induction period. It would be helpful to mention the media used  for example,  MSC differentiation medium used during the process, including any supplements or factors that are crucial for inducing pluripotency or differentiation into multiple germ layers. This would provide readers with a comprehensive understanding of the conditions required to generate functional MSCs.Additionally, to strengthen the claims of regenerative potential of exosome, further assays to assess the regenerative capabilities of exosomes derived from iMSCs should be included.

Regarding the figures, Figures 1C & D, Figure 2D, Figure 3D, and Figures 4A & C appear to be blurred and lack the resolution necessary for clear interpretation. These figures are crucial for supporting the results and should be reprocessed or replaced with higher-quality images to ensure clarity and improve the overall visual presentation. Enhanced clarity in these figures would make the data more convincing and allow for a better understanding of the experimental outcomes."

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Dr Mahmood S Choudhery, PhD

Academic Editor

PLOS ONE

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Additional Editor Comments:

The section under this heading “Generation of Embryoid Bodies from iMSCs (EB-iMSCs)” should be divided into two distinct parts to improve clarity and flow. The second part should focus on the protocol for generating embryoid bodies (EBs) from induced Mesenchymal Stem Cells (iMSCs), including a detailed step-by-step process for embryoid body formation. This would include key factors such as cell seeding densities, culture conditions, and the duration of the induction period. It would be helpful to mention the media used for example, MSC differentiation medium used during the process, including any supplements or factors that are crucial for inducing pluripotency or differentiation into multiple germ layers. This would provide readers with a comprehensive understanding of the conditions required to generate functional MSCs.Additionally, to strengthen the claims of regenerative potential of exosome, further assays to assess the regenerative capabilities of exosomes derived from iMSCs should be included.

Regarding the figures, Figures 1C & D, Figure 2D, Figure 3D, and Figures 4A & C appear to be blurred and lack the resolution necessary for clear interpretation. These figures are crucial for supporting the results and should be reprocessed or replaced with higher-quality images to ensure clarity and improve the overall visual presentation. Enhanced clarity in these figures would make the data more convincing and allow for a better understanding of the experimental outcomes.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Nashwan et al., have performed a comparative analysis of EVs isolated from IMSCs and ADMSCs. The authors need to work on the following comments to improve the manuscript.

1. Authors have demonstrated the role of EVs specifically in terms of mesenchymal cells and the generation of EVs from immune cells has not been discussed. Therefore, the following references are suggested to be cited in the third paragraph after reference #12

a. PMID: 36570199

b. PMID: 36713536

c. PMID: 38753658

2. Authors are suggested to incorporate these studies, which have indicated the negative role of MSC in bone cancer formation, along with reference 6.

a. PMID: 36769180

3. Authors are suggested to explain the findings of senescence data, if there is a significant difference between the SFM and ADMS-EVs in apoptosis and MTT data then why is it not a case of senescence?

4. The discussion section is again solely dedicated to mesenchymal cells, it should be broadly discussed in terms of EVs.

Reviewer #2: Ababneh and colleagues have explored the potential of extracellular vesicles (EVs) derived from induced mesenchymal stem cells (iMSCs) as an alternative to EVs from adipose-derived mesenchymal stem cells (ADMSCs) for regenerative medicine. The study compares iMSC-EVs and ADMSC-EVs in terms of size, effects on cell viability, apoptosis, migration, and senescence in vitro culture conditions. The authors argue that iMSC-EVs demonstrate a larger particle size and superior performance in enhancing cell viability and migration, while showing comparable effects on senescence and apoptosis reduction to ADMSC-EVs. Despite its innovative premise, the manuscript suffers from methodological inconsistencies, insufficient data to support key claims, unclear controls, and low-quality figure presentation. The manuscript thus requires major revisions to improve clarity, address limitations, and provide additional data where needed. These changes will strengthen the manuscript and enhance its overall impact.

Specific Comments:

1) EV regenerative properties: The main goal of the paper was to prove regenerative and wound healing properties of EVs derived from induced MSCs. However, it is not clear if these properties are general for any EVs. It would be thus necessary to include a comparison with control EVs (for example from Human Dermal Fibroblasts used in the experiments) that should lack wound-healing properties to establish the specificity of the observed effects.

2) Mis-cited references: The introduction lacks adequate references to support the claims. Mis-cited and irrelevant references make it difficult to verify key statements. Ensure that all references are accurate, recent, and contextually relevant. Here are a couple of examples for consideration:

a) Introduction, 3rd paragraph. The reference #13 does not support the statement that EVs are superior than MSCs. “EVs derived from human MSCs (hMSCs) have demonstrated superiority in maintaining similar functions to MSCs and avoiding apparent adverse effects [13].”

b) The reference #14 does not contain any mention of EVs. “Moreover, MSC-derived EVs (MSC-EVs) can replace intact MSCs in tissue repair and regeneration [14].”

c) Address inconsistencies in reference style (e.g., "(Van Niel, D’Angelo and Raposo, 2018)" vs. numbered citations).

3) EV dose used: The description of EV concentrations (10–50 µg/mL) provided in the methods section is not consistent with the results. Specify which concentration was used for each figure and experiment.

4) Figure quality and resolution: All figures need substantial improvement in quality. Microscopy images (Figures 1C, 1D, 5A, 5C) are unclear and need higher resolution. Axes, labels, and legends (e.g., Figure 4B) must be readable and clearly described.

5) EV size inconsistency: Discuss the inconsistency and heterogeneity in iMSC-EV size profiles observed in Figure 2C.

6) Data absent: Please provide data supporting the following statement for Figure 1. “Additionally, all iMSC samples were negative for the MSC- negative markers (CD34, CD45, CD14, CD11b, CD79a, CD19, and HLA-DR).”

7) Spontaneous apoptosis: Address the reason for high apoptosis levels in Figure 4C and clarify if the media conditions (e.g., absence of serum) caused this effect. Explore alternative controls like EVs derived from human dermal fibroblasts that do not protect from apoptosis.

8) Figure 3 description: Inconsistent description of experiment. Unclear if EVs or cells were stained with Dil.

a) In methods “iMSCEVs, MSCs-EVs, and PBS were labelled with DiI fluorescent dye selectively stained the plasma membrane and lipids.” Whereas in Results “To assess if HDFs can internalize EVs, cells were incubated with Dil as a fluorescent dye and a lipophilic stain, integrated into lipid bilayer membranes, and emitted orange-red fluorescence”.

b) The merged image of ADMSC-EVs does not show the red-Dil labeling staining.

c) The following statement lacks quantitative data for support. Please do quantitative image analysis to provide stronger proof. “The internalization efficiency of iMSC-EVs was higher than that of ADMSC-EVs.”

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Reviewer #1: Yes:  NAMRATA ANAND

Reviewer #2: No

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Comparative Analysis of Extracellular Vesicles from Induced and Adipose-Derived Mesenchymal Stem Cells: Implications for Regenerative Medicine

PONE-D-24-47284R1

Dear Dr. Ababneh,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mahmood S Choudhery, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors are suggested to check the reference style all over the manuscript aftrer implementing the studies and make it according to the journal recomendations.

Reviewer #3: The authors have made the necessary corrections by adhering to the reviewers' opinions.

The authors addressed the Reviewers' requests carefully and corrected them.

The authors enriched the Manuscript.

The work is valuable and can significantly contribute to the scientific community in regenerative applications.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: Yes:  NAMRATA ANAND

Reviewer #3: No

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