Mesenchymal Stromal Cells and Extracellular Vesicles: A Novel Therapeutic Paradigm for Mitochondrial Dysfunctions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript provided an overview of the latest studies on the effects of MSCs and MSC-EVs on mitochondrial diseases from the following sections: the genetics of mitochondrial diseases, diagnosis of mitochondrial diseases, treatment and prognosis of mitochondrial diseases, therapeutic potential of mesenchymal stromal cells and their extracellular vesicles in mitochondrial diseases, challenges and prospects of MSC- and MSC-EV-based therapies for mitochondrial diseases. however, several aspects require substantial clarification and refinement before publication.

1. The reference list is disorganized and not arranged in numerical order.
2. Please diversify the use of references to support related arguments, rather than repeatedly citing the same few sources. For example, References [1] and [16] have been cited multiple times throughout the text.
3. Several sections of the manuscript lack supporting references, weakening the foundation and credibility of the arguments presented. For example, line 56-58, line 107-114, line 222-229, line 304-307, line 359-364, line 388-401.
4. The presentation of Part 2 requires substantial revision. At present, this section contains an extensive list of information that appears more descriptive than analytical, which disrupts the logical flow of the manuscript. To improve clarity and conciseness, summarize and synthesize the key points in this section, rather than presenting them as a straightforward enumeration. For instance, similar to the effective presentation in Table 1, organizing the main content of this part into a table or a structured summary would enhance readability and help readers grasp the core information more efficiently.
5. The manuscript currently devotes substantial text to introducing the genetics of primary mitochondrial diseases (PMD) in the earlier sections, yet the subsequent discussion on research progress regarding MSCs and MSC-EVs in mitochondrial diseases appears to focus predominantly on secondary mitochondrial diseases (SMD). This imbalance may lead to a disconnect between the genetic background presented and the therapeutic context discussed.
6. The description of the treatment and prognosis of mitochondrial diseases is currently limited to a single paragraph (line 222-233), which may not sufficiently cover this critical aspect of the topic.
7. Section 5.1 discusses the therapeutic potential of mesenchymal stromal cells in mitochondrial Diseases; however, the claims made in this part are not sufficiently supported by references.
8. The authors provided an overview of the latest in vitro and in vivo studies elucidating the mechanisms by which MSCs and MSC-EVs act in mitochondrial diseases.
9. The discussion on the role of MSCs and MSC-EVs in mitochondrial diseases in this manuscript focuses predominantly on evidence from cellular and rodent models. While valuable, the text also acknowledges that the exact mechanisms underlying these effects remain unclear. Given this limited and still-evolving mechanistic understanding, the claim that MSCs and MSC-EVs represent a "novel therapeutic paradigm" for mitochondrial diseases appears premature.
10. While the authors provide a comprehensive overview of both MSCs and MSC- MSC-EVs in mitochondrial diseases, which one is more clinically promising and why.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript presents a timely and clinically relevant narrative review summarizing current evidence on the potential of MSCs and their extracellular vesicles to restore mitochondrial function. The topic is important given the limited therapeutic options for mitochondrial diseases. The review is generally well organized and includes a useful synthesis of in vitro and in vivo studies.
However, the paper would benefit from sharper conceptual framing and stronger critical stratification of evidence, particularly regarding the distinction between primary mitochondrial diseases and secondary mitochondrial dysfunction, which currently blurs the scope and may overstate translational readiness.
Major issues:
1. The manuscript uses the term “mitochondrial diseases” broadly, but much of the MSC-EV literature summarized is focused on conditions characterized by mitochondrial dysfunction, rather than genetically defined PMDs. This is acknowledged by the authors, but the framing still implies a stronger evidence base for PMD than currently exists. Consider revising the title, or explicitly separate the review into two main pillars: PMD evidence (limited; mostly MSCs) vs SMD evidence (broader; MSCs and EVs).
2. The review promotes MSC-EVs as an attractive alternative to whole-cell therapy (low immunogenicity, scalability), but the authors state that no studies have evaluated MSC-EVs in primary mitochondrial diseases to date. Maybe include a short dedicated paragraph (or a boxed summary) clarifying what is known in PMD, what is known only in SMD, which claims are extrapolations and should be framed as hypotheses.
3. The manuscript highlights mitochondrial transfer via TNTs/gap junctions and EV-mediated cargo delivery as mechanisms of rescue. However, the paper also acknowledges significant mechanistic uncertainties regarding the fate and durability of donated mitochondria, integration into mitochondrial networks, and whether effects are transient or stable. 
4. Table 1 provides a strong overview, but several entries lack key details (e.g., dosage/route “not specified”), which limits interpretability and comparison across studies. 

Minor issues:
1. Introduction is informative and includes prevalence estimates and clinical relevance, however, the objectives could be stated more directly and earlier. 
2. Figure 2 is helpful in contrasting MSC vs MSC-EV mechanisms (whole mitochondria vs molecular cargo), but the figure caption and text could be tightened to avoid implying a level of certainty that is not fully supported mechanistically. 
3. Some sections transition too quickly from broad MSC applications (e.g., osteoarthritis, diabetes) to mitochondrial disease framing; consider trimming or explicitly justifying their inclusion as SMD examples. 
4. The “Challenges and Prospects” section is one of the strongest parts of the paper and could be moved slightly earlier or summarized in a boxed “key limitations” paragraph for emphasis. 

Conclusion:
This review has potential to be a useful contribution; however, substantial revisions are required to better align the scope and claims with the strength of available evidence, especially for primary mitochondrial diseases. Strengthening methodological transparency and improving critical stratification of PMD vs SMD evidence would significantly improve rigor and clarity. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This review manuscript outlines the potential of mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) in repairing mitochondrial dysfunction diseases. It covers the genetic background of mitochondrial diseases, the difficulties in clinical diagnosis, the biological mechanisms of MSCs and EVs, and relevant in vitro and in vivo research data. The authors point out that MSC-EVs offer the possibility of cell-free therapy.

Comments:
1. Although the manuscript emphasizes discussing "mitochondrial diseases (MDs)," the experimental evidence and animal models are almost entirely from "secondary mitochondrial dysfunction" (such as ischemic injury, diabetes, kidney injury, etc.), with limited coverage of hereditary primary diseases (such as MELAS, LHON). Is there currently experimental evidence for the efficacy of MSC-EVs against PMDs? Could unpublished studies have been overlooked?

2. This manuscript points out that MSCs can perform mitochondrial transfer and mentions the therapeutic potential of MSC-EVs, but it does not specifically distinguish between effects originating from the cell itself and effects achieved solely by EVs. For example, it is not yet confirmed whether mitochondrial transfer can be achieved via EVs, rather than requiring direct cell contact or passage through channels (such as tunneling nanotubes). The authors could consider adding a discussion on whether EVs can independently deliver mitochondria, comparing this to the mechanism of direct cell transfer.

3. While this manuscript extensively covers in vivo/in vitro experiments, it does not systematically compile current clinical trial data for MSC/EV treatment of mitochondrial diseases, and lacks analysis of its efficacy or side effects. For example, supplementing with current clinical research data from clinicaltrials.gov, or comparing the therapeutic effects of different EV sources (bone marrow, umbilical cord, adipose tissue).

4. This manuscript mentions the low immunogenicity of EVs, but does not further analyze whether EVs induce adverse reactions in animal models, especially in cases of repeated infusions or high doses. The authors could further discuss existing reports of EV-induced immune activation, coagulation problems, or clearance rates, enriching the safety assessment.

5. Although MSCs can transfer to healthy mitochondria, their persistence, integration, and replication capabilities do not appear to be fully resolved. Does this mean that this therapy is only a short-term supportive treatment?

6. Are the studies in Table 1 all studies related to mitochondrial diseases, or just a few representative studies?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in present form

Reviewer 2 Report

Comments and Suggestions for Authors

In my opinion the  article should be published in its current form.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made appropriate revisions according to the reviewers' comments.