PONE-D-21-10675

Isolation and long-term expansion of murine epidermal stem-like cells

PLOS ONE

Dear Dr. Xia,

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PLOS ONE

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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

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

Reviewer #1: 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: Yes

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4. 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

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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: The manuscript describes an incremental development of protocols to culture murine primary keratinocytes. The experiments used to indicate that a variant of KFSM medium containing 0.06mM Ca2+ permits long term expansion of primary murine keratinocytes are robust and the data do support the idea of long term culture. Reporting conditions for long term culture alone are not novel since other publications such as Hager et al Journal of Investigative Dermatology, 1999,112(6):971-976, have described similar findings, however the authors of this current manuscript have extended the transcriptomic analysis of the keratinocytes subjected to their culture conditions.

That said, it is unusual for primary cells to undergo at least 70 passages so it would have been useful if the authors could have provided an explanation for this enhanced proliferative capacity. Their data imply a degree of stem cell like characteristics in the cell population they generate (eg CD34, CD200, expression) but they have not indicated why such stem cells have this proliferative capacity. It would be useful to measure telomere lengths, expression of mTert and activity of the murine telomerase holoenzyme complex in addition to markers of cell senescence such as expression of p16 INK4a. Moreover, they do not provide analyses of the karyotype at early and late passage numbers. These additional data could be supportive of a putative epidermal stem cell identity especially since the doubling time of the cells grown in low calcium KSFM seems to decrease substantially after passage 20 (figure 2C). This could of course imply the selection of a more highly proliferative cell population but it could also imply cell transformation

Another drawback of the data provided is that there seems to be no attempt to correlate transcriptomic identity of the cell population to passage number. Given that doubling times decrease, it may be assumed that the transcriptome of the bulk population does change so taking a “snapshot” of gene expression does not provide an accurate description of a potentially changeable phenotypic environment. This potential change with increasing passage number would need to be quantified before the method can be confirmed to produce larger numbers of murine keratinocytes that are useful for studying epidermal biology and disease as stated in the discussion section. If data from these studies can be provided, the findings may be of interest to the readership of PLOSone

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Reviewer #1: No

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Isolation and long-term expansion of murine epidermal stem-like cells

PONE-D-21-10675R1

Dear Dr. Xia,

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,

Majlinda Lako

Academic Editor

PLOS ONE

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

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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

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

Reviewer #1: 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.

Reviewer #1: 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

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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)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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