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    Review Commons Reviews for Refereed Preprint RC-2021-00693

    Ferrari G. et al., DLL4 and PDGF-BB regulate migration of human iPSC-derived skeletal myogenic progenitors.

    __Reviewer #1 (Evidence, reproducibility and clarity): __ Summary:

    The paper presented by Ferrari et al., aims to improve the migration capacity of hiPSC- derived myogenic progenitors. For this purpose, the authors used a previously published well characterized hiMPs model and focussed on the modulation of NOTCH and PDGF signaling pathways. The rational to target these pathways was based on muscle cells migrations molecular events observed during developmental described in the literature.

    __Major comments: __ Are the key conclusions convincing?

    This is a very interesting paper. Few clarifications as suggested below need to be done before being fully convincing. Enrichment test and heat maps and the network analysis are not well explained in terms of which genes were selected and why, and in terms of which gene set were selected and why. In some cases, the information may be given in the paper, but it is not easy for the reader to find it. It should be stated more clearly. For example, in Fig2C why these eight were chosen for the heat maps and why not other genes known to be involved in myogenesis, cell migration etc. Similar comment for figure 3 A, D and G. Another example, in Fig 2E, on what basis are some gene sets chosen to be shown in this figure when there are many more significant in the supplementary table.

    We thank the Reviewer for their positive feedback and for this comment. Although some answers to the queries could be found within the figure legends, we agree that figures could have been more self-explanatory, and we will amend them accordingly. We will also add additional information into the main text to clarify those specific points.

    In response to the specific queries:

    • All enrichment heat maps were generated from GO lists or KEGG pathways.
    • 2C: these were chosen instead of other myogenic or cell migration markers for consistency with our previous study (Figure 2C in Gerli et al Stem Cell Reports 2019).
    • 3A, D, G: details of the GO lists used to generate heat maps were available in the relative figure legend.
    • 2E: enrichment pathways – we listed pathways shared between at least 2 of the three groups and with relevance to cellular migration.

    Figure 4F is impossible to interpret without a clear description of how the subnetwork is extracted, was a list of gene list submitted to string, if so which genes and why? Secondly, why are there many nodes with no edges? Is it all of the nodes that are in that GO-Term, if so it needs to be clarified? Was this the most strongly deregulated go-Term according to string analysis?

    We thank the Reviewer for this comment. This specific GO list was selected for its highly relevant title/topic, i.e.: “positive regulation of cell migration”. Details on this point could also be found in the specific figure legend, where we specified how the network is extracted and constructed. There are several nodes with no edges as the edges represent predicted functional association and therefore, a lack of edges suggests a lack of interaction.

    Figure 4 B, C, D and E: (1) The authors should clarify what figure 4B is? Is 1,2,3,4 different time point? Treated or untreated cells?

    We apologise with the Reviewer for not having provided enough information on this point. 1,2,3 and 4 are four sequential time points of untreated cells. We will amend the figure to make this clearer.

    (2) Figure C: Is the graph showing the cell distribution of both treated and untreated cells? If yes is it possible to give a different shape for the control cells and see if indeed more control green shape would be observed in this plot? (In the supplementary data there is the distribution showing the treated v untreated, but the clusters are not visible)

    We thank the Reviewer for this helpful comment. We agree that this will increase the quality of the figure. We will distinguish treated and control cells within figure 4C by replacing dots with different shapes for treated and untreated samples.

    (3) Would it be possible to take some of the parameters in Figure 4D and show the distribution in treated vs untreated and perform the statistical analysis? (eg is there a significant difference for the parameter total distance between control and treated?). Or, may be just show some of the results in figure S4C and E in the main text.

    We thank the Reviewer for this comment. We agree that it will be better to move S4C into the main figure and we will action this point in the revised version of the manuscript.

    (4) Why pooling the 3 independent experiment together? Looking at the data in Figure S4, it seems that one treated sample is very similar to the control, thus weakening the conclusion. The replicates in this figure are biological replicates. Yet the papers present 4/5 different cell lines, so why only 3 of them are used here? Is there some explanation regarding the outsider (cell line age, number of division etc). Might be worth adding data from the other cell lines (1 or 2 more).

    We thank the Reviewer for this point. The experiment shown in figure S4E has been performed with one cell line (N5) and independent experimental replicates were assessed for the statistical analysis. We are not sure why there appears to be an outlier in some cases, and this is why it was important to replicate this experiment three times. However, we will also repeat this experiment with another cell line applying more stringent conditions to strengthen this point.

    (5) Figure 4 H and I: What are the statistic actually comparing: treated v untreated for each cell lines or different cell lines against each other? If the former, then how is it possible to have a 139 fold change with such a weak p value of 0.042? If the latter, then why is a p-value given for each of the 3 cell lines? Also, the number and source of replicates is unclear - N=3 is stated, so was each cell line done in triplicate? If so, how many fields per replicate?

    We are happy to clarify this point for the Reviewer. The statistical analysis compares treated vs. untreated samples within the same genotype. The high fold change observed is likely due to the large standard deviation of the dataset, which was also highlighted as raw data in the figure panel (bottom part of each picture in white colour font). For this reason, we have repeated this experiment multiple times and validated it across three independent cell lines.

    Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. It would important to also show the migratory capacity of these cells in vivo.

    Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. Human muscle cells engraftment and tracking in immunodeficient mice could be easily done. Engrafted muscle can be harvested 2-3 weeks after engraftment, and measurement of the distance from the engraftment point could be done (Site of injection could be labelled with tattoo die). This would be a month/month and half of work. Immunodeficient mice would cost around £1500 (n=6 mice per group => total of 12 mice) plus the cost of housing.

    Are the data and the methods presented in such a way that they can be reproduced? Are the experiments adequately replicated and statistical analysis adequate? See comments in first paragraph. The authors should probably be able to answer easily to the different concerns raised above.

    We thank the Reviewer for these comments. We agree that the suggested in vivo experiment might strengthen our work and we are currently sourcing all required materials to perform it. Additionally, we will perform a similar, quasi-vivo, experiment to study migration in a species-specific setting by delivering cells in 3D models in vitro (e.g. Maffioletti SM et al., Cell Reports 2018). This strategy will provide a solid alternative to the in vivo assay, in the eventuality that the xenogeneic setting will limit the resolution of the proposed transplantation experiment.

    Minor comments: typo "Onthology" should be "Ontology" in figure 2E. Some of the data in Figure S4E should be moved to the main text.

    Thank you for highlighting these minor comments. We will correct the typo and move data from figure S4 into the main figure 4.

    Reviewer #2 (Evidence, reproducibility and clarity): In this manuscript, Ferrari and colleagues provide solid data indicating that the Notch ligand DLL4 and PDGF-BB regulate the migration of myogenic progenitors derived from human pluripotent stem cells (PSC). These studies built from recent work by the same group (Gerli et al, Stem Cell Reports, 12:461, 2019), in which the authors documented that Notch and PDGF-BB signaling enhances migration and expression of stem cell markers while inducing perivascular cell features in muscle satellite cells. Here the authors perform similar in vitro studies in PSC-derived myogenic progenitors and conclude that the same effect is observed in this population of cells. The results are clear and well presented.

    Throughout the manuscript, the authors emphasize the importance of such findings for the future therapeutic application of a PSC-based therapy to treat patients with muscular dystrophy since multiples skeletal muscles need to be targeted in this group of diseases. Unfortunately, the authors do not provide transplantation data. The results would be highly meaningful if they show that observed in vitro changes (transcriptomes and chamber assay) result in meaningful migration in vivo using the systemic delivery, but as it is, the data do not support the claims and conclusions.

    We thank Reviewer 2 for their comments. We were pleased to read that they found our study and data solid, clear and well presented. Although we agree with the Reviewer that in vivo evidence would strengthen our findings, we would like to highlight that our study did not aim to be a translational investigation of the therapeutic potential of treated hPSC derivatives for muscle cell therapy (we believe our manuscript’s title reflects this). We see this work more as a foundational study to establish the required evidence for future, follow up transplantation studies focused on the therapeutic potential of this approach (something requiring a dedicated project, funding and months/years of work).

    Moreover, we believe that xenogeneic transplants are of limited use to investigate a complex species-specific phenomenon such as transendothelial cell migration. For this very reason we moved back to intraspecific transplantsin past studies (e.g.. Tedesco et al Sci Transl Med 2012). However, as a key aim of our study is to obtain data specific to human cells and given that we already performed mouse-in-mouse in vivo intra-arterial delivery experiments using DLL4 and PDGFBB treated primary cells in Gerli et al. Stem Cell Reports 2018, we are therefore proposing and planning to:

    • Test transendothelial migration with another quasi-vivo microfluidic assay orthogonal to the reported transwell experiments. This will model intraspecific (i.e., human-in-human) transendothelial migration under flow conditions.
    • Assess evidence of migration in human 3D muscles setting up a novel invasion assay in our in vitro 3D muscle models.
    • Perform intramuscular delivery of treated vs. untreated cells as per Reviewer 1 request to assess migration in skeletal muscle in vivo. This approach will optimise in vivo experiments in a 3Rs compliant fashion, avoiding invasive procedures in animals to study intravascular delivery.

    Reviewer #2 (Significance): Significance is limited if only in vitro data are provided. However if the authors are able to show enhanced engraftment upon systemic transplantation of human PSC-derived myogenic progenitors upon DLL4 and PDGF-BB treatment, the significance would be high.

    Please see our reply to the previous point.

    In terms of existing literature, there are publications reporting systemic delivery of murine PSC-derived myogenic progenitors as well as transcriptome and in vitro migration studies. It would probably be appropriate to cite them.

    We apologies to the Reviewer for this oversight. We will add the following papers which include systemic delivery of murine PSC-derived myogenic progenitors as well as transcriptome and migration studies: Matthias N et al., Exp Cell Res 2015; Incitti T et al., PNAS 2019.

    If systemic engraftment is observed, the manuscript would be of interest to the skeletal muscle and stem cell biology/regenerative medicine community.

    Please see our reply to the initial point.

    __Reviewer #3 __(Evidence, reproducibility and clarity):

    In this manuscript, the authors exploited the signal-mediated activation of NOTCH and PDGF pathways, by one week-long delivery of DLL4 and PDGF-BB to cultures of hiPSC-derived myogenic progenitors in vitro, to improve their migration ability. They performed transcriptomic and functional analyses across human and mouse primary muscle stem cells and human hiPSC-derived myoblasts, including genetically corrected hiPSC derivatives, to show that DLL4 and PDGF-BB treatment modulates pathways involved in cell migration, including enhanced trans-endothelial migration in transwell assays.

    The increased migratory ability, and in particular enhancing extravasation, is a fundamental property required for optimal performance of hiPSC myogenic derivatives, upon their intravascular delivery; hence, the finding reported here are of extremely high potential interest in term of solution of one of the major bottle-neck of cell therapy. However, there are important issues that need to be resolved by the authors with additional experimentation, that I recommend performing, in order to improve this manuscript.

    We sincerely thank the Reviewer for acknowledging the extremely high relevance and potential of our paper for muscle gene and cell therapies and for providing constructive feedback to improve our manuscript.

    1. The most critical issue here is that the authors fail to provide evidence that DLL4/PDGF-BB-treated cultures of hiPSC-derived myogenic progenitors do not lose their myogenic potential and are able to form myotubes, upon interruption of treatment. It would be also important to determine when (how many days after withdrawal of DLL4/PDGF-BB) the full myogenic properties of these cells are recovered. From the RNAseq datasets shown by the authors, it appears that DLL4/PDGF-BB-treated hiPSC-derived myoblasts do not express the key genes of myogenic identity (MyoD) and early differentiation (myogenin), while expressing genes of mesenchymal/vessel-derived lineages. It is imperative that the authors show that these changes are reversible, upon withdrawal of DLL4/PDGF-BB. This should be show by an unbiased transcriptomic analysis (RNAseq) of hiPSC-derived myoblasts after withdrawal of DLL4/PDGF-BB, that should be integrated with functional evidence showing that these cells can resume their ability to form differentiated myotubes, upon exposure to myogenic culture cues in vitro.

    We thank the Reviewer for this comment. We agree that this is an important and feasible experiment which will add important information to our work. We performed similar work in our previous study and already observed phenotype reversion of treated cells upon release of the stimuli within a few passages in cultures. However, we agree that this requires systematic assessment and quantification. To this aim, we will assess the reversibility of the DLL4 & PDGF-BB effect by stopping treatment at day 7 and then assessing skeletal myogenic differentiation capacity of target cells at sequential passages and time points post-treatment. Analysis of the differentiation index at different time points will provide functional evidence on the myogenic potential of hiPSC-derived myogenic progenitors post-withdrawal of DLL4 & PDGF-BB. We believe that the Reviewer’s suggestion for transcriptomic analysis via RNA-seq might be overly costly for the purpose of identifying the myogenic potential of treated cells post-withdrawal of treatment, and that qPCR panels alongside immunofluorescence staining may be sufficient.

    1. A parallel evidence in vivo should be also provided, showing that DLL4/PDGF-BB-treated hiPSC-derived myoblasts do not express MyoD and myogenin when delivered intravascularly, but regain their expression after they have crossed the vessel endothelium and have entered the skeletal muscles.

    We thank the Reviewer for suggesting this experiment. We agree that this would be a very interesting point to address; however, it might be very challenging to address this question with the proposed in vivo experiment. Nonetheless, we believe that with a combination of in vitro and in vivo assays we will be able to satisfactorily answer the question: Do DLL4 and PDGF-BB-treated myogenic progenitors re-gain myogenic potential upon entering skeletal muscle tissue? To this aim, we aim to analyse muscles following intramuscular transplantation of treated and untreated cells. Moreover, to model intra-vascular delivery and have high resolution imaging, we aim to adapt a microfluidic platform to perform trans-endothelial assays and selectively differentiate cells that successfully cross the blood vessel layer. Although likely to be very challenging, we will attempt to capture or stain those very cells in order to assess the expression of myogenic markers as requested by the Reviewer.

    If these experiments could firmly demonstrate that DLL4/PDGF-BB-treatment reversibly promotes migratory properties of hiPSC-derived myoblasts (as predicted, but not demonstrated in previous works from the same group, using mouse or human primary muscle stem cells - Cappellari et al. 2013; Gerli et al. 2019), then this work could be a great interest in term of basic and translational biology and clearly suitable for publication in a top journal.

    We thank the Reviewer for this constructive feedback and for seeing the great potential of our work in terms of basic and translational biology. We assume there was a typo in the sentence in brackets with a missing “as” (“..not demonstrated as in previous work...”): we indeed demonstrated the effect of DLL4 and PDGFBB in vivo extensively in our previous work.

    Other points:

    • Fig. 2A. it looks like there are some outlier RNAseq sample replicates that might negatively impact at the statistical level on the subsequent analysis. This issue is likely due to the heterogeneity of the samples (both untreated and treated) and could be resolved by replacing outlier samples with new replicates.

    We thank the Reviewer for this comment. Although we agree that replacing those samples with new replicates might improve our statistical analyses, this will be financially challenging at this stage and perhaps also not completely reflecting the real variability of the experimental setup.

    • Along the same line as above, sample heterogeneity following treatment might be resolved by a better understanding of optimal doses of DLL4/PDGF-BB and time of exposure, which I recommend the authors to define by additional experiments.

    We thank the Reviewer for this comment. This is a potentially interesting experiment, which we have not performed as we took advantage of previous knowledge and dose-response on primary mouse and human myoblasts. Overall, we believe that this experiment might not be strictly required at this stage, given that we have already solid evidence of response in hiMPs with a defined concentration and exposure time of DLL4 and PDGFBB.

    Reviewer #3 (Significance):

    If these experiments could firmly demonstrate that DLL4/PDGF-BB-treatment reversibly promotes migratory properties of hiPSC-derived myoblasts (as predicted, but not demonstrated in previous works from the same group, using mouse or human primary muscle stem cells - Cappellari et al. 2013; Gerli et al. 2019), then this work could be a great interest in term of basic and translational biology and clearly suitable for publication in a top journal and could be interesting for a wide audience in regenerative medicine.

    We thank the Reviewer once again for this constructive feedback and for seeing the great potential of our work in terms of basic and translational biology, as well as for regenerative medicine.

    Please note that the following statement will be added to the Acknowledgements section of our revised manuscript: "For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust (FC001002)".

    Once again, we sincerely thank all Reviewers for their positive, constructive and insightful comments, which motivate us to further improve our work. We also thank the Review Commons editorial team for guidance and assistance.

    Prof. Francesco Saverio Tedesco, University College London and The Francis Crick Institute, London, UK.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors exploited the signal-mediated activation of NOTCH and PDGF pathways, by one week-long delivery of DLL4 and PDGF-BB to cultures of hiPSC-derived myogenic progenitors in vitro, to improve their migration ability. They performed transcriptomic and functional analyses across human and mouse primary muscle stem cells and human hiPSC-derived myoblasts, including genetically corrected hiPSC derivatives, to show that DLL4 and PDGF-BB treatment modulates pathways involved in cell migration, including enhanced trans-endothelial migration in transwell assays. The increased migratory ability, and in particular enhancing extravasation, is a fundamental property required for optimal performance of hiPSC myogenic derivatives, upon their intravascular delivery; hence, the finding reported here are of extremely high potential interest in term of solution of one of the major bottle-neck of cell therapy. However, there are important issues that need to be resolved by the authors with additional experimentation, that I recommend performimg, in order to improve this manuscript.

    1. The most critical issue here is that the authors fail to provide evidence that DLL4/PDGF-BB-treated cultures of hiPSC-derived myogenic progenitors do not lose their myogenic potential and are able to form myotubes, upon interruption of treatment. It would be also important to determine when (how many days after withdrawal of DLL4/PDGF-BB) the full myogenic properties of these cells are recovered. From the RNAseq datasets shown by the authors, it appears that DLL4/PDGF-BB-treated hiPSC-derived myoblasts do not express the key genes of myogenic identity (MyoD) and early differentiation (myogenin), while expressing genes of mesenchymal/vessel-derived lineages. It is imperative that the authors show that these changes are reversible, upon withdrawal of DLL4/PDGF-BB. This should be show by an unbiased transcriptomic analysis (RNAseq) of hiPSC-derived myoblasts after withdrawal of DLL4/PDGF-BB, that should be integrated with functional evidence showing that these cells can resume their ability to form differentiated myotubes, upon exposure to myogenic culture cues in vitro.

    2. A parallel evidence in vivo should be also provided, showing that DLL4/PDGF-BB-treated hiPSC-derived myoblasts do not express MyoD and myogenin when delivered intravascularly, but regain their expression after they have crossed the vessel endothelium and have entered the skeletal muscles. If these experiments could firmly demonstrate that DLL4/PDGF-BB-treatment reversibly promotes migratory properties of hiPSC-derived myoblasts (as predicted, but not demonstrated in previous works from the same group, using mouse or human primary muscle stem cells - Cappellari et al. 2013; Gerli et al. 2019), then this work could be a great interest in term of basic and translational biology and clearly suitable for publication in a top journal.

    Other points:

    • Fig. 2A. it looks like there are some outlier RNAseq sample replicates that might negatively impact at the statistical level on the subsequent analysis. This issue is likely due to the heterogeneity of the samples (both untreated and treated) and could be resolved by replacing outlier samples with new replicates.
    • Along the same line as above, sample heterogeneity following treatment might be resolved by a better understanding of optimal doses of DLL4/PDGF-BB and time of exposure, which I recommend the authors to define by additional experiments.

    Significance

    If these experiments could firmly demonstrate that DLL4/PDGF-BB-treatment reversibly promotes migratory properties of hiPSC-derived myoblasts (as predicted, but not demonstrated in previous works from the same group, using mouse or human primary muscle stem cells - Cappellari et al. 2013; Gerli et al. 2019), then this work could be a great interest in term of basic and translational biology and clearly suitable for publication in a top journal and could be interesting for a wide audience in regenerative medicine.

    Expertise of this reviewer:

    Muscle regeneration; Muscular Dystrophies; Signaling and Epigenetics

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Ferrari and colleagues provide solid data indicating that the Notch ligand DLL4 and PDGF-BB regulate the migration of myogenic progenitors derived from human pluripotent stem cells (PSC). These studies built from recent work by the same group (Gerli et al, Stem Cell Reports, 12:461, 2019), in which the authors documented that Notch and PDGF-BB signaling enhances migration and expression of stem cell markers while inducing perivascular cell features in muscle satellite cells. Here the authors perform similar in vitro studies in PSC-derived myogenic progenitors and conclude that the same effect is observed in this population of cells. The results are clear and well presented.

    Throughout the manuscript, the authors emphasize the importance of such findings for the future therapeutic application of a PSC-based therapy to treat patients with muscular dystrophy since multiples skeletal muscles need to be targeted in this group of diseases. Unfortunately, the authors do not provide transplantation data. The results would be highly meaningful if they show that observed in vitro changes (transcriptomes and chamber assay) result in meaningful migration in vivo using the systemic delivery, but as it is, the data do not support the claims and conclusions.

    Significance

    Significance is limited if only in vitro data are provided. However if the authors are able to show enhanced engraftment upon systemic transplantation of human PSC-derived myogenic progenitors upon DLL4 and PDGF-BB treatment, the significance would be high.

    In terms of existing literature, there are publications reporting systemic delivery of murine PSC-derived myogenic progenitors as well as transcriptome and in vitro migration studies. It would probably be appropriate to cite them.

    If systemic engraftment is observed, the manuscript would be of interest to the skeletal muscle and stem cell biology/regenerative medicine community.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The paper presented by Ferrari et al., aims to improve the migration capacity of hiPSC- derived myogenic progenitors. For this purpose, the authors used a previously published well characterized hiMPs model and focussed on the modulation of NOTCH and PDGF signaling pathways. The rational to target these pathways was based on muscle cells migrations molecular events observed during developmental described in the literature.

    Major comments:

    • Are the key conclusions convincing? This is a very interesting paper. Few clarifications as suggested below need to be done before being fully convincing. Enrichment test and heat maps and the network analysis are not well explained in terms of which genes were selected and why, and in terms of which gene set were selected and why. In some cases, the information may be given in the paper, but it is not easy for the reader to find it. It should be stated more clearly. For example, in Fig2C why these eight were chosen for the heat maps and why not other genes known to be involved in myogenesis, cell migration etc. Similar comment for figure 3 A, D and G. Another example, in Fig 2E, on what basis are some gene sets chosen to be shown in this figure when there are many more significant in the supplementary table. Figure 4F is impossible to interpret without a clear description of how the subnetwork is extracted, was a list of gene list submitted to string, if so which genes and why? Secondly, why are there many nodes with no edges? Is it all of the nodes that are in that GO-Term, if so it needs to be clarified? Was this the most strongly deregulated go-Term according to string analysis? Figure 4 B, C, D and E:

    (1) The authors should clarify what figure 4B is? Is 1,2,3,4 different time point? Treated or untreated cells?

    (2) Figure C: Is the graph showing the cell distribution of both treated and untreated cells? If yes is it possible to give a different shape for the control cells and see if indeed more control green shape would be observed in this plot? (In the supplementary data there is the distribution showing the treated v untreated, but the clusters are not visible)

    (3) Would it be possible to take some of the parameters in Figure 4D and show the distribution in treated vs untreated and perform the statistical analysis? (eg is there a significant difference for the parameter total distance between control and treated?). Or, may be just show some of the results in figure S4C and E in the main text.

    (4) Why pooling the 3 independent experiment together? Looking at the data in Figure S4, it seems that one treated sample is very similar to the control, thus weakening the conclusion. The replicates in this figure are biological replicates. Yet the papers present 4/5 different cell lines, so why only 3 of them are used here? Is there some explanation regarding the outsider (cell line age, number of division etc). Might be worth adding data from the other cell lines (1 or 2 more).

    (5) Figure 4 H and I: What are the statistic actually comparing: treated v untreated for each cell lines or different cell lines against each other? If the former, then how is it possible to have a 139 fold change with such a weak p value of 0.042? If the latter, then why is a p-value given for each of the 3 cell lines? Also, the number and source of replicates is unclear - N=3 is stated, so was each cell line done in triplicate? If so, how many fields per replicate?

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. It would important to also show the migratory capacity of these cells in vivo. -Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. Human muscle cells engraftment and tracking in immunodeficient mice could be easily done. Engrafted muscle can be harvested 2-3 weeks after engraftment, and measurement of the distance from the engraftment point could be done (Site of injection could be labelled with tattoo die). This would be a month/month and half of work. Immunodeficient mice would cost around £1500 (n=6 mice per group => total of 12 mice) plus the cost of housing.
    • Are the data and the methods presented in such a way that they can be reproduced? Are the experiments adequately replicated and statistical analysis adequate?

    See comments in first paragraph. The authors should probably be able to answer easily to the different concerns raised above.

    Minor comments:

    typo "Onthology" should be "Ontology" in figure 2E. Some of the data in Figure S4E should be moved to the main text.

    Significance

    Describe the nature and significance of the advance, existing literature, audience: Generating iPSC cell lines with an improved capacity to migrate will be of high interest for the neuromuscular field, and could be a potential therapeutic strategy applicable for many neuromuscular disorders.

    Muscle cell engraftment is quite challenging as the capacity of these cells to populate different muscles is very poor. Improving the cell migration, survival and proliferation may thus help to improve the muscle cell engraftment strategy.

    Expertise:

    I have an expertise in neuromuscular disorders, muscle stem cells (human and murine, in vitro and in vivo), as well as an expertise in omics analysis.

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