Dear Dr. Ytterdal,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

The manuscript "The neural transdifferentiation potential of bone marrow mesenchymal stem cells" is an interesting investigation in the field of stem cell therapeutics. But it needs significant improvement before acceptance. So, revise the manuscript in the light of reviewers' comments.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: No

Reviewer #2: Yes

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

Reviewer #1: No

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. 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??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The study by Ytterdal et al entitled “The neural transdifferentiation potential of bone marrow mesenchymal stem cells” examines the ability of mesenchymal stem cells (MSCs) to transdifferentiate to a neuro and glial lineages. The study addresses a decades-long controversy regarding the ability of MSCs to transdifferentiate from mesodermal to neuroectodermal lineage, a phenomenon suggested to be due to phenotypic plasticity and experimental artifact rather than true lineage conversion. While the current study offers some important insights in terms of timing and proteomic changes during transdifferentiation, there remains many deficits in the study including a lack of quantitation and statistical analysis that limit the overall interpretation that acquisition of a neural/glial phenotype by MSCs represents transdifferentiation.

1) Introduction lines 77-82: The authors cite 3 clinical trials using MSCs in MS. The first 2 trials represent IV administration of MSCs in RRMS patients. The limitations of the 2 studies should be noted, including IV administration on a RRMS patient population that already benefit from highly effective therapies thus limiting the need for MSCs in this population. A description of Ref 17 should also be included in this paragraph because it represents IT administration and reported efficacy in a progressive patient population.

2) Include additional demographics on the six donors including MS subtype, EDSS, disease duration, gender, and age.

3) Figure 1 is subjective and could easily represent a biased interpretation.

a. The undifferentiated MSCs which were repeatedly passaged over the 3 weeks are shown at subconfluence whereas the differentiated MSCs are confluent. The morphology of representative confluent MSCs should be used to compare.

b. The advantage of using 6 separate MSC lines is to provide quantitative data. Any changes in morphology needs to be demonstrated for all 6 lines. The authors should apply high resolution imaging metrics using Image J or Cell Profiling to provide more robust and unbiased quantitation of basic morphological differences between MSCs, N- and G-MSCs from all 6 lines. At the very least, additional representative images of the other 5 lines should be included in the supplemental data. Figure legend should state that the images are representative.

c. The authors should address the morphological similarity between neural and glial differentiated cells despite claiming they represent different lineages. Other than being “different” from MSCs, please comment on how N or G MSC morphology compares to brain derived neural/glial cultures.

4) Figure 2 scratch assay:

a. Please justify the conclusion that the scratch assay is reflective of migratory capacity of MSCs. Clearly the proliferation rate of N- and G-MSCs is reduced and thus reflected in the reduced rate of closure in the scratch assay. The limitation of this assay in terms of assessing migratory capacity and its relationship to proliferation should be clearly delineated.

b. The discussion point line 454 “decreased migratory capacity could hinder homing to CNS lesions” should be removed or revised since migration in this context refers to specific chemotactic responses that are typically measured in a trans well assay.

c. The legend states average and SE from 3 different experiments. Please clarify whether the graph represents all 6 cell lines that were tested in 3 different experiments and how the average and SE were determined. Data from all 6 cells lines needs to be included along with statistical analysis to support any differences between MSCs and N/G-MSCs.

5) Figure 3 proliferation: See point 4c above regarding data representing all 6 cells lines and statistical analysis.

6) Figure 4 needs to include quantitative analysis of immunostaining in all 6 cell lines to demonstrate reproducibility. The figure showing a representative image of each marker is meaningless without quantitative analysis and statistics. There appears to be more GFAP staining in N/G MSCs but there are also more cells in the images. This reflects the fact that immunofluorescence is not ideal to quantitate protein expression and should be supported by a more quantitative method (Western blotting) in all 6 lines.

7) Figure 5, the PCA plot appears to represent 2 samples of MSCs, 2 samples of N and G MSCs at 7 days, and 3 samples of N and G MSCs at 14 and 21 days. Please clarify whether these are representative of only 1 cell line in duplicate/triplicate or 3 cell lines? Please clarify whether or not proteomics was performed in all 6 lines.

8) Figure 6

a. please clarify why there are 7-9 biological replicates when the methods states 6 separate cell lines and the PCA plot only shows 2-3.

b. What were the criteria for selecting the markers represented in the heatmap?

c. The proteomic data should be analyzed by performing unbiased enrichment analysis to identify pathways that are altered. Without pathway analysis, it’s not clear that the markers as shown were cherry picked or are meaningful representatives of cell proliferation or differentiation towards a certain lineage.

9) Figures 7, 8 and 9

a. The markers in these figures were from a predefined panel. Please explain the criteria for selection of the panel. Why are some of the markers not included in Figure 6?

b. Clarify, again, whether the averages in the graphs represent 1 or 3 or 6 separate cell lines (see comment to Figure 5).

c. If not representative of all 6 cell lines, then additional protein quantitation should be performed (i.e. western blot) to confirm these protein changes in all 6 lines.

d. Please perform statistical analysis on graphs in figures 7, 8 and 9.

10) Figure 9, the oligodendroglial markers chosen do not accurately represent oligodendrocyte lineages. PDGFA is not typically expressed in oligodendrocytes as it is the ligand for oligo marker and receptor PDGFRA. The authors should show more typical markers of oligodendrocytes such as olig1 and olig2.

11) Discussion

a. line 388 concludes that the expression patterns showed high consistency with minimal donor to donor variability. This statement is not supported by the data in all 6 cell lines as outlined in the above comments.

b. Line 433, when discussing a more CNS-relevant secretory profile, only one of the factors (PDGFA) is secreted the others are cytoplasmic or membrane associated. Please revise this paragraph to clarify how these specific proteins might be related to improved trophic support of differentiated MSCs.

12) A major question regarding transdifferentiation is whether or not it represents an in vitro artifact due to stress caused by altered culture conditions. StemPro MSC medium contains a growth supplement (serum alternative). How much of the results shown here in terms of proliferation, ‘migration’, and expression of proliferation markers are due to removal of the growth supplement when culturing in StemPro NSC media? Furthermore, is the N or G phenotype reversible, meaning that after 3 weeks if you put the cells back in StemPro MSC do they resume proliferation? An even more important question is whether or not mesodermal differentiation (adipocytes, osteocytes, chondrocytes) is lost after N or G differentiation. Without additional experiments to answer these questions, the discussion needs to include a more balanced and tempered interpretation of the current findings regarding transdifferentiation.

13) Address why G and N MSCs have similar morphology and protein expression despite exposure to different growth factors. The appropriate control would be StemPro NSC media without growth factors to show that the growth factors are indeed promoting differentiation into glial and neural lineages. Without this experiment, the possible alternative interpretations of this data should be included.

14) Include a more thorough discussion on the controversy around transdifferentiation of MSCs and the limitations of the current study. Multiple references from the 2000’s initiated the controversy underlying whether transdifferentiation occurs or is an in vitro artifact. Those references are omitted here but serve important perspective on the topic. Please discuss the limitations of the current study including the lack of functional data, lack of evidence of terminal differentiation, and lack of differentiation controls for proliferative effects of the culture media as outlined above. Furthermore, the overall lack of evidence in the literature supporting in vivo differentiation of MSCs or Neural MSCs into neural lineages upon transplantation is a major limitation to the interpretation that neural differentiation is relevant to the regenerative capacity of these cells.

Reviewer #2: The manuscript titled “The neural transdifferentiation potential of bone marrow mesenchymal stem cells” reports original research investigating the ability of MSCs from multiple sclerosis (MS) patients to acquire neural and glial-like characteristics in vitro. The study is well designed, technically sound, and supported by detailed proteomic analysis that enhances its scientific depth. The findings are consistent and reproducible, indicating early neuro-glial lineage commitment. Overall, the paper is clearly written and contributes meaningful molecular insight into MSC biology. However, several issues concerning methodological transparency, statistical rigor, and ethical reporting should be addressed before it can be considered for publication. Please find the detailed comments below.

Major Comments

1- The introduction does not sufficiently explain the scientific reasoning for selecting MSCs from MS patients rather than healthy donors. The rationale should clarify whether the intent was to model disease-specific MSC behavior, to evaluate the feasibility of autologous transplantation in MS, or to test whether the disease state influences differentiation potential. Without this context, the choice of donor source appears arbitrary and limits interpretation of the findings.

2- A section describing statistical methodology is missing. The results include qualitative claims of “significant” differences in migration and proliferation, but no information is provided on statistical tests, sample size, or p-values.

3- The fluorescence images are interpreted qualitatively. If possible, quantitative analysis (e.g., mean fluorescence intensity or proportion of marker-positive cells across replicates) should be included.

4- More detailed information about the six MS donors is needed, including sex distribution, MS subtype (progressive vs. relapsing), disease duration, and treatment history. These factors are relevant for assessing potential biological variability and for enabling reproducibility.

5- The manuscript lists “N/A” under Ethics Statement, although human-derived cells were used. Please revise to include the appropriate ethical approval (REK#159326) and confirm that written informed consent was obtained under the SMART-MS protocol.

6- The proteomic dataset is extensive, but the analysis is limited to marker-level observations. Incorporating pathway-level bioinformatic analyses (e.g., Gene Ontology or KEGG enrichment) would help identify broader biological processes and signaling pathways associated with transdifferentiation. This can be performed using the existing dataset and would considerably enrich the study’s interpretive depth.

7- Terminology: “Transdifferentiation”. Since functional neural or glial activity was not demonstrated, it would be more accurate to refer to the observed changes as lineage priming or phenotypic conversion rather than full transdifferentiation.

Minor Comments

1- Several typographical errors should be corrected (e.g., “trans-differentiated,” “neurodegeneratice”).

2- Confirm that data files in the Supporting Information fully correspond to the figures and text.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes:Violaine K. HarrisViolaine K. HarrisViolaine K. HarrisViolaine K. Harris

Reviewer #2: Yes:Niyaz Al-SharabiNiyaz Al-SharabiNiyaz Al-SharabiNiyaz Al-Sharabi

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Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Mar 25 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols....

We look forward to receiving your revised manuscript.

Kind regards,

Vikash Chandra, PhD

Academic Editor

PLOS One

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Additional Editor Comments:

Dear Authors,

Although most of the reviewers’ comments have been adequately addressed, the following concerns still require attention:

1. The newly added graphs in Figure 4 are very small and difficult to read. Please revise the figure to improve clarity and readability.

2. Please comment on whether SOX2, PDGF, and NEFL were also observed to be upregulated in the proteomic screen.

3. Line 448: The sentence beginning with “After 3 weeks of …” is incomplete, as the word “transdifferentiation” is missing.

Please revise the manuscript in light of the above comments and resubmit your revised manuscript for further consideration.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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.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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: The authors have adequately addressed my comments and the manuscript is acceptable for publication. A few suggested minor edits that do not require another round of review.

1) The new graphs in Figure 4 are very small and difficult to read.

2) Suggestion that the authors comment on whether SOX2, PDGF, and NEFL were also observed to be upregulated in the proteomic screen.

3) Line 448: After 3 weeks of .... (transdifferentiation is missing)

Reviewer #2: Thanks to the authors for providing the revised version of the manuscript entitled “The neural transdifferentiation potential of bone marrow mesenchymal stem cells.” The authors have addressed all major and minor comments carefully and thoroughly. The rationale for using MSCs from MS patients is now clear, the statistical framework has been strengthened, and the quantitative analysis of imaging and proteomic data has improved the rigor of the study. The added donor information and corrected ethics statement further enhance transparency and reproducibility. Clarification of terminology and inclusion of pathway-level analyses have also brought the interpretation into closer alignment with the data.

The revised manuscript now presents a coherent and well-supported study.

**********

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To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

The neural transdifferentiation potential of bone marrow mesenchymal stem cells

PONE-D-25-55478R2

Dear Dr. Ytterdal,

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,

Vikash Chandra, PhD

Academic Editor

PLOS One