Differentiation of mouse embryonic stem cells into cells with spermatogonia-like morphology with chemical intervention-dependent increased gene expression of LIM homeobox 1 (Lhx1)

  1. Cameron Moshfegh
  2. Sebastian G. Rambow
  3. Seraina A. Domenig
  4. Aldona Pieńkowska-Schelling
  5. Ulrich Bleul
  6. Viola Vogel

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Abstract

No abstract available

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  1. Version published to 10.1016/j.scr.2022.102780
  2. Version published to 10.1101/2021.10.12.464060v2 on bioRxiv
  3. Version published to 10.1101/2021.10.12.464060v3 on bioRxiv
  4. PeerRef

    Discussion, revision and decision

    Discussion and Revision

    Author

    Reviewer 1 (Takehiko Ogawa, Yokohama City University, ogawa@yokohama-cu.ac.jp ):

    We had already suggested in the "Limitations" section on page 10 of the manuscript-PDF testis transplantation experiments to test biological functionality for future studies: https://www.biorxiv.org/content/10.1101/2021.10.12.464060v1.full.pdf

    However, these experiments will require new cell lines, new funding, more resources, more logistics, and new ethics approval which we are considering for future studies. We believe that the observations described in our manuscript are valuable to the scientific community and are a basis to conduct further studies.

    Reviewer 2 (Dr. Pradeep G Kumar, Rajiv Gandhi Centre for Biotechnology, and kumarp@rgcb.res.in )

    There are no line numbers in the manuscript-PDF but the pages are numbered: https://www.biorxiv.org/content/10.1101/2021.10.12.464060v1.full.pdf Since the pages of the manuscript-PDF are numbered, we think that citation of the manuscript is unproblematic. The text of the manuscript has been checked by multiple experienced authors and found not to require the suggested changes.

    Reviewer (Takehiko Ogawa)

    I think that authors admit the limit of the study, which I think is serious. I think the paper has its own value with limitation as almost always in most cases. I would like to take “verified with reservation” as my final decision.

    Decision

    Takehiko Ogawa: Verified with reservations

    Pradeep G Kumar: Verified manuscript

    Verified with reservations

  5. PeerRef

    Peer review report

    Reviewer: Dr. Pradeep G Kumar Institution: Rajiv Gandhi Centre for Biotechnology email: kumarp@rgcb.res.in

    Section 1 – Serious concerns

    • Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
    • Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

    ####Section 2 – Language quality

    • How would you rate the English language quality? High quality

    Section 3 – validity and reproducibility

    • Does the work cite relevant and sufficient literature? Yes
    • Is the study design appropriate and are the methods used valid? Yes
    • Are the methods documented and analysis provided so that the study can be replicated? Yes
    • Is the source data that underlies the result available so that the study can be replicated? Yes
    • Is the statistical analysis and its interpretation appropriate? Not applicable
    • Is quality of the figures and tables satisfactory? Yes
    • Are the conclusions adequately supported by the results? Yes
    • Are there any objective errors or fundamental flaws that make the research invalid? No

    Section 4 – Suggestions

    • In your opinion how could the author improve the study?

    • Do you have any other feedback or comments for the Author?

    Text requires improvement at some places (citing them is difficult as pages and lines are not numbered). Some examples are given below:

    .......was associated with molecular markers of the PGC...... (was associated with the expression of molecular???)

    Primordial germ cells (PGCs), the developmentally first founder cells of the germline, are induced from epiblast cells on around embryonic day (E)6.25 by external signals,

    Furthermore, it was shown for that male mouse ES cells cultured in serum that subjected to a chemical intervention, including a timed exposure to a combination of the a SIRT1 inhibitor Ex-527, the a DNA methyltransferase inhibitor RG-108 and the an electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with induced the expression of molecular markers of the PGC

    Section 5 – Decision

    Verified manuscript

  6. PeerRef

    Peer review report

    Reviewer: Takehiko Ogawa Institution: Yokohama City University email: ogawa@yokohama-cu.ac.jp

    Section 1 – Serious concerns

    • Do you have any serious concerns about the manuscript such as fraud, plagiarism, unethical or unsafe practices? No
    • Have authors’ provided the necessary ethics approval (from authors’ institution or an ethics committee)? not applicable

    Section 2 – Language quality

    • How would you rate the English language quality? High quality

    Section 3 – validity and reproducibility

    • Does the work cite relevant and sufficient literature? Yes
    • Is the study design appropriate and are the methods used valid? Almost Yes
    • Are the methods documented and analysis provided so that the study can be replicated? Almost Yes
    • Is the source data that underlies the result available so that the study can be replicated? I think, Yes
    • Is the statistical analysis and its interpretation appropriate? Not applicable
    • Is quality of the figures and tables satisfactory? Almost Yes
    • Are the conclusions adequately supported by the results? No
    • Are there any objective errors or fundamental flaws that make the research invalid? Please describe these thoroughly.

    I would like to request authors to test the validity of cells with spermatogonia-like morphology (CSM) as spermatogenic stem cells. For this, transplantation of CSM into the seminiferous tubules in the testis of recipient mice is necessary. This may be an additional work, but authenticity of CSM as SSCs cannot be claimed without such experiment. They may claim that in case of ES cells most experiments do not perform blastocyst injection experiment to confirm the pluripotency of ES cells. However, culture system for ES cells has a long history and was established as robust method to maintain their pluripotency. It is actually the 2iL system as shown in this study as well. In case of SSCs or GS cells, the culture system is still fragile in my understanding, compared to the 2iL. The spermatogenic ability that SSCs and GS cells have could be lost during the cultivation in many cases. In particular, chemical intervention shown in this study could disturb cell’s intrinsic system, which could make almost anything happen. The enhanced expression of Lhx1 also could be an aberrant result of such turmoil in the cells. I do not insist that is the case, but intentionally taking a position to be extremely skeptical to the results. In order to dispel such doubts, it is necessary to do the transplantation experiments and prove that the CSM maintained the spermatogenic ability as SSCs.

    Section 4 – Suggestions

    • In your opinion how could the author improve the study?

    Readers would wonder what the CSM is exactly. This naming means that CSM could be SSCs but possibly not, although they look like SSCs. It was honest naming but confusing in what the CSM really is. In order to start the experiment with CSM, authors should clear such ambiguous point. Thus, I recommend a transplantation experiment as stated above.

    • Do you have any other feedback or comments for the Author?

    In Page 6, it is described that medium change twice daily with a third of the volume at hour 8 and 16 was critical for the appearance of CSM. In case of the medium change with half of the volume every 12 hours, it was written that the CSM did not appeared at all. Authors argued that endogenously produced soluble factors might be the cause. I wonder what extent of difference could be there between the two method of culture medium change, regarding to the concentration of endogenously produced soluble factors. A simulation of the change in concentration of such hypothetical substances might help the speculation.

    Section 5 – Decision

    Requires revisions

  7. Version published to 10.1101/2021.10.12.464060v1 on bioRxiv