PONE-D-24-20935Stem cell culture conditions affect in vitro differentiation potential and efficiency of mouse gastruloid developmentPLOS ONE

Dear Dr. Blotenburg,

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.

You should try to address all of the Reviewers' points detailed below, through experiments or by added detail, clarification or discussion.It is particularly important that you establish the reproducibility of your findings through analysis of at least one additional mouse ES cell line, from a distinct genetic background. It is also critical that you include detailed experimental descriptions, including considerations of cell density, molar concentrations of growth factors, sources of media and additives, as requested by the Reviewers. Also provide more detail in terms of the design of your scRNA-seq experiments. Please include time-dependent analysis of key regulators during gastruloid formation, e.g. by immunostaining or ISH, as a means of linking lineage priming bias in 2D cultures and differential lineage representation in the gastruloids.  

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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?

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: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: No

Reviewer #2: Yes

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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: In the manuscript entitled “Stem cell culture conditions affect in vitro differentiation potential and efficiency of mouse gastruloid development” Blotenburg and colleagues confirmed and extended previous observations that the cell culture conditions, and specifically the pluripotency state of the stem cell population, affect gastruloid development in term of morphology, elongation, and cell type composition. By performing ScRNA-seq analysis of mESCs and gastruloids the authors identify the pathways that control the efficiency of gastruloid formation. The results are interesting and provide insights into the complexity of stem cell plasticity. However, in my opinion, there are important points that need to be addressed prior to publication.

Major points:

The impact of different culture conditions on gastruloids formation have been previously reported. Unfortunately, the authors incorrectly cite the relevant literature. For instance, Cermola et al (Stem Cell Report 2021, ref.31) reported the effect of ESCs grown in different culture conditions//pluripotency state including but not limited to i) DMEM/FBS 15%/LIF, ii) N2B27 2i/LIF, and FGF/Activin -induced EpiLC and EpiSCs on gastruloids formation. They demonstrated that culture conditions significantly impact gastruloid formation, affecting i) cell aggregation (size of cell aggregates at 48 hours), ii) elongation (proportion of correctly elongated gastruloids), and iii) morphology (presence of aberrant gastruloids/multiple protrusions).

The authors should discuss their findings more thoroughly in the context of the current literature and properly cite the relevant papers. This will not diminish the significance of their studies.

Materials and Methods

- Pag. 4, line 84: PBS0 and TrypLE are not defined. Please specify

- Pag. 5, lines 92-93: the concentration of 2i inhibitors (CHIR and PD) are expressed as %, but it is not clear % of what mass/vol?. Please clarify or indicate the molar concentration.

- Pag. 7, line 147: the sentence ‘Cells with either less than 500 reads and 100 genes detected in less than 2 cells were filtered out’. Please rephrase or clarify

Results

Pag. 8 lines 195-196: the authors claim that ..”pluripotency states in between naïve and ground state can be produced with short-term pulses of 2i and ESLIF”. However, this conclusion lacks experimental evidence. The authors should provide at least some characterization of the cells defined as “intermediate” states of pluripotency (conditions named 3, 4, 5 and 6) between conditions 1 and 2.

Figure 1, pre-culture conditions, including the number of cells plated/density, the type of plates and coating used, are not specified.

Figure 1, panel A’. At the high cell density, assessing the effect of culture conditions on cell colony morphology seems difficult to evaluate/quantify. Therefore, the phenotype-based analysis of the intermediate states proposed here lacks convincing evidence. On the other hand, a molecular-based approach, i.e. the identification of specific markers for the 6 conditions, for instance based on data from the RNAseq analysis, seems not sufficient per se (see below).

Pag. 9 line 204. …”cells from all pre-culture conditions except ‘condition 2’ generate gastruloids efficiently”. The 2i medium is routinely used to capture naïve pluripotency, and so, the observation that ESCs grown in 2i medium (condition 2) are unable to elongate is unexpected. It is likely that 2i ESCs are ‘locked” in the naïve pluripotent state and that this eventually delay/prevent exit from pluripotency and symmetry breaking. Indeed, condition 5, that is 48h in LIF medium is sufficient to rescue the elongation efficiency (see also Discussion section below). Please, discuss.

Figure 1, the values of the elongation index are overall lower than those previously reported by different laboratories (2.5 or higher), and the abnormal value (see Fig.1, panel D, conditions 3, 4, 6 replicate B) appears to correspond to the diameter rather than the length. Indeed, the diameter is larger than expected (100-200 microM). Likely, it is due to the use of the largest circle (from Max Inscribed Circles Fiji plug-in) as the gastruloid diameter.

Pag. 10 line 242-243, ScRNAseq analysis (Figure 2) revealed that ESCs even if grown in 6 different conditions, display only two different transcriptional and epigenetic signatures. These results raised some issues:

a.- What about the intermediates states of pluripotency. Is the ScRNAseq analysis able to discriminate the cells grown in the 6 conditions? The cells pre-grown in the 6 different conditions display significant differences in their transcriptome? Please clarify

b. Is the transcriptome profile relevant for the gastruloid formation efficiency?. For instance, cells from conditions 1, 5, and 6 seems to segregate together from the seq analysis but display a completely different gastruloid formation efficiency (Figure 1).

It seems that the presence of LIF for the last 24 hour (compare conditions 1, 5, and 6) is sufficient to induce a similar signature/cell identity (transcriptome and epigenetic signature).

Pag. 13 line 323 to Pag. 14 line 346, ScRNAseq analysis (Figure 3) revealed that gastruloids generated with cells grown under different conditions (1, 5, and 6), have a different lineage compositions. These interesting results raise some issues:

a.- Is the composition of the gastruloids analyzed here (mainly condition in line with that reported in the literature with respect to types/number of cell lines identified, etc.?

b.- the high fraction of mesoderm in gastruloids derived from conditions 5 and 6 correlates with the induction of mesodermal markers in ESCs grown under these conditions?

Discussion

Pag. 14, lines 362-364. …”While in our study 2i grown mESCs yielded the lowest efficiency of gastruloid elongation, another study using E14 mESCs reported increased efficiency of elongation after mESCs were maintained in 2i medium. (31)”. This is not correct. In the paper by Cermola et al. (ref 31 ms), mESCs were maintained (passaged) in DMEM/FBS 15%/LIF not in 2i. Then, to generate gastruloids, mESCs were seeded at a low density (300 cells/cm²) on gelatin-coated plates and cultured in N2B27 2i/LIF for 5 days. It is important to note that the 2i medium also includes LIF (2iLIF). Under these culture conditions, 90%–95% of ESC colonies show a domed round-domed shaped phenotype. Besides the difference in the culture conditions, other technical differences in the protocol need to be consider:

1) mESCs were dissociated with a milder accutase treatment, and not trypsin

2) The protocol includes a FACS sorting step to eliminate dead cells and cellular debris prior to aggregation.

Thus, the culture conditions and the protocols used in the paper by Cermola et al. (ref. 31) and those reported herein are different and should be considered. These important differences should be addressed and discussed, and the sentence should be rephrased accordingly.

Reviewer #2: The study of Blotenburg et al. addresses whether stem cell culture conditions, particularly the introduction of 2i, affect the development of mouse gastruloids and whether an optimised mESC pluripotency state would provide more consistent results. These are relevant questions because the gastruloid model system is widely used and, contrary to the 3D/N2B27 part, 2D culture conditions lack a defined protocol and have been shown to vary substantially (sometimes even in the same study).

Throughout the manuscript the authors present results suggesting that modulation of the pluripotency state of mESCs significantly affects the phenotype (morphology), transcriptome and epigenome of gastruloids, ultimately impacting the specification of certain cell fates. Although these results are interesting, they were obtained from only one mouse cell line. Also, the same occurs for the optimisation work carried out to improve the reproducibility/consistency of the gastruloid system. Checking the ‘universality’ of their findings in at least two more mESCs (also used by other labs working with gastruloids) would significantly improve the work and its significance to the community. Another aspect that requires further work regards the control of cell number during 2D culture. According to the manuscript, cells were split every second day in a 1:5 ratio during the modulation of their pluripotency state. Did the authors examine whether cell number was kept stable across the different conditions? If the numbers changed, they should check whether seeding density alone affects the pluripotency state and, further down, gastruloid formation. It is important to isolate/control the seeding density variable during these experiments to understand the 2i effects more accurately.

After modulating the 2D culture conditions, Blotenburg et al. developed mouse gastruloids according to the standard protocol and performed a detailed morphological analysis. Here, it would be nice if the authors pointed out the morphological differences they see in the gastruloid images of Figure S1A (e.g. “ridge in the centre and protrusions at the posterior end”). The authors then tested the effects of a temporal shift in the CHIR treatment in the various culture conditions. However, it is not possible to correctly interpret this experiment without any Brachyury stainings at 48, 72 and 96h. Also, Blotenburg et al. should perform the read-out of condition 2 at 144h; there is published data indicating that gastruloids whose cells have been treated with 2i only achieve their maximum elongation at that time (e.g. Beccari et al., 2018). Finally, the authors should also revise terms like “ ‘perceptiveness’ to the CHIR treatment” and explain the meaning of “ ‘efficient’ gastruloid formation”.

In the last part of the manuscript, the authors address the transcriptome and epigenome comparison they did for the different culture conditions, both before and post gastruloid formation, and conclude that there are significant differences in their molecular signatures. Here the authors must revise their annotations and try to be more accurate in defining the different pluripotency states and cell types present in both the pre-culture and gastruloid datasets. For defining the different pluripotent cell states, the authors should use terminology, supported by gene references, that have already been published in other studies (e.g. Nichols and Smith, 2009, Smith 2017, Morgani et al., 2017 and Cermola et al., 2021). Regarding the gastruloid data, Blotenburg et al. should expand the number of genes used for annotation (e.g. Tbx1, Tcf15, Sox17, Noto, Pax6, Otx2, etc.) and use correct anatomical/embryological terminology (e.g. ‘Caudal epiblast’ instead of ‘Tailbud’, ‘Spinal cord’ instead of ‘Neurons’, etc.). Importantly, the authors also need to look at genes that can provide some information about developmental time as it is critical to discard heterochrony; UMAPs showing the integration of the different gastruloid datasets with that of embryos would be very helpful as well. Additionally, the authors should include in the materials and methods section information about the number of cells analysed per condition during the previous single cell experiments.

One of the key results of this manuscript is the finding that modulation of pre-culture conditions, with the addition of 2i, seems to have a strong effect later on cell fate specification during gastruloid formation, particularly in the amount of neural and mesodermal tissues. This finding contrasts with what is reported in the preprint of Rosen et al., 2022, which also finds a bias in the amount of neural and mesodermal cells but links it to inter-gastruloid heterogeneity; Blotenburg et al. should discuss this issue.

Overall, the main question addressed in this manuscript is pertinent and the experiments are interesting. However, there is a considerable amount of work to be done in order for the results to be accurate and significant to the community.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-20935R1Stem cell culture conditions affect in vitro differentiation potential and mouse gastruloid formationPLOS ONE

Dear Dr. Blotenburg, 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.

Both Reviewers and I are appreciative of the amount of time and effort put into this extensive revision. As you will see from the comments below, they are mostly positive, but there remain some concerns about the significance of the data presented, which I am keen for you to address to some extent. However, I am keen to keep revisions within the scope of the original reviews and requests for clarification or validation.

It is my view that the results presented are important for comparability of mouse gastruloid protocols and experimental results, and have been strengthened by the revisions. It is equally important to anchor the results on objective and transferable reference markers.

In that regard, I would like the Authors to address 2 remaining major points raised by Reviewer 2.

1. Discuss the implications for mouse gastruloid formation of long-term 2i culture conditions for ES cell maintenance.

2. Perform Brachyury staining at 48-72h to assist in the deconvoluting the impact of ES culture conditions and the timing of CHI pulsing in the differences observed at 120h.

Also, please make sure that you systematically include references for pathways, cell and tissue annotations.

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Please submit your revised manuscript by Nov 16 2024 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.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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 https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Cristina Pina, MD DPhil

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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 #2: (No Response)

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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 #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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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 #2: 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 #2: Yes

**********

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: (No Response)

Reviewer #2: I appreciate the authors' efforts in conducting some of the additional experiments and analyses I suggested, particularly the evaluation of the impact of distinct culture conditions on gastruloid morphology using different cell lines. These new results and the corresponding revisions have enhanced the manuscript, providing some clarity to the study. However, some issues are still unresolved and further work is required before the manuscript is ready for publication.

Main issues:

- The transcriptomic and epigenetic investigations remain limited to one cell line. Given the high variability found between cell lines in the initial culture conditions, it is important to extend these analyses to more cell lines and provide a thorough discussion of the results. In this discussion, the authors should also explain why longer periods of 2i treatment - recommended by laboratories such as that of Austin Smith to achieve a ‘tabula rasa’ state - were not considered for this study.

- The manuscript still lacks sufficient evidence showing whether the different culture conditions alter the cell state of the gastruloids at 48 hours (before CHIR treatment), and if this causes the observed differences at 120 hours. It is critical to provide this data to interpret the CHIR timing shift correctly. For instance, is symmetry breaking delayed when 2i is used? The rationale for altering the CHIR treatment timing needs to be clearly explained, with evidence. As previously suggested, stainings for key primitive streak markers like Brachyury between 48 and 72 hours would be a solution.

Minor issues:

- It should be visually clear which results in Figure 1 correspond to which cell line. The same applies to the new figures.

- The data suggesting the involvement of Shh signalling (“ectodermal priming signature with the expression of Gibx2, Ptch1, Sox11, and Lefty1, indicative of Sonic Hedgehog (Shh) signalling”) is insufficient and lacks appropriate references. Additionally, the role of this signalling in pluripotency should be discussed.

- The authors’ annotation of the different cell types and pluripotency states has improved but still needs refinement. For example, is Tbx4 a mesodermal marker? I suggest the authors review each marker mentioned in the text and provide proper references.

- The issue of potential heterochrony should be resolved by including a heatmap showing the expression of Hox genes across the different conditions.

- The authors should lower their claims regarding the novelty of their screening approach as similar methodologies have been published already by other laboratories (e.g. Lutolf’s lab).

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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: No

Reviewer #2: No

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Stem cell culture conditions affect in vitro differentiation potential and mouse gastruloid formation

PONE-D-24-20935R2

Dear Dr. Blotenburg,

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.

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Kind regards,

Cristina Pina, MD DPhil

Academic Editor

PLOS ONE