PONE-D-21-14310

Whole embryonic detection of maternal microchimeric cells highlights significant differences in their numbers among individuals

PLOS ONE

Dear Dr.Naoki Irie ,

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 address all issues raised by both reviewers, and in particular, authors must demonstrate that microchimeric cells are all of maternal origin as GFP+ cells can also come from another fetus of the same litter carrying the maternal GFP.

Please submit your revised manuscript by Oct 15 2021 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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PLOS ONE

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Reviewers' comments:

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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: N/A

Reviewer #2: I Don't Know

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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: No

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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: The authors present a whole embryonic detection method for maternal microchimeric (MMc) cells using transgenic mice to test whether the number of maternal cells differs from one embryo to another. They justify their method of detection on the whole embryo instead of testing separated organs and tissues, as they hypothesize that if MMc does not differ in number from one embryo to another the overall number of MMc cells in the fetus may not differ among the different individuals.

And they further explain that different numbers of MMc cells have been reported among various tissues in previous studies and could reflect a particular accumulation in a specific tissue. They therefore want to overcome this by testing the entire embryo.

For this purpose, authors have crossed female mice heterozygous for the GFP locus (GFP+/-, from F1 BALB/c x C57BL/6-GFP) with non-GFP BALBc male mice, and offspring with no GFP gene (GFP-/-) were analyzed for GFP+/- MMc cells.

The major inconvenient with this system is that fetuses GFP-/- may have received microchimeric cells GFP+/- from other fetuses from the same litter. Therefore authors cannot assert that GFP-/+ cells are maternal.

Moreover they observed that “MMc” differed in frequency among individual fetuses.

The authors need to provide information about the other fetuses from the same litter.

Were there more GFP + fetuses in the litter where one of them was very positive?

How many fetuses from the same litter were analyzed?

It is very difficult to discuss these results until we have this information.

To absolutely demonstrate that maternal cells are maternal cells and not cells from other fetuses I would suggest to construct another model of MMc detection. Considering that this is technically feasible, one way out might be to realize the same experiences with mothers being heterozygous for Green and Red fluorescence, thus MMc would be GFP+/- RFP+/- among either heterozygous GFP+/- RFP -/- embryos or among heterozygous GFP-/- RFP +/- embryos.

Finally, a second important point: although this is a count of GFP + cells on a whole embryo, since the detection technique is done by FACS, it is a pity that other cell markers are not added to determine the phenotype. A multiparameter analysis would give additional indications as to the type of Mc cells.

The study, nevertheless very interesting, must be reconsidered by the authors.

Reviewer #2: In the manuscript “Whole embryonic detection of maternal microchimeric cells highlights significant differences in their numbers among individuals” the authors introduce a technique to examine the quantity of allogeneic cells in a murine model. Utilizing a breeding strategy to produce GFP- embryos in a GFP+ mother, the authors were able to utilize GFP+ cell presence in the embryos as an indicator of maternal microchimerism. To measure the quantity of GFP+ cells in the embryos they implemented a fluorescence-activated cell sorting strategy to filter cells on PI staining (for live cell detection) and GFP (for allogeneic cell detection). The presence of GFP+ cells, presumably from the mother, were identified in several of the embryos with one presenting with a notably larger proportion of GFP+ cells. This technique provides a useful tool for the analysis of maternal microchimerism in embryo development and may help to answer some of the enigmas of maternal microchimerism. This is an interesting report on a technique that can aid researchers in the understanding of fetomaternal chimerism and mechanisms in utero. I found the methods to be generally well described and took particular interest in the study design. I also have some comments for the authors to consider for further improvements in clarity and dissemination of their findings:

Major comments:

1. The sample size of the study is not clear from the text or figures. Samples sizes listed in lines 196-198 suggest a smaller number of samples compared to figure 3B. It would be helpful to clearly indicate how many litters and embryos were studied at each gestational age for the experiments.

2. The results section should include either a detailed section or table containing descriptive statistics for each gestational age group. As the study focus is in the prevalence and proportion of GFP cells at different gestational ages, I would suggest this to include data on GFP cell proportions, measure of variation, proportion of embryos with detectable GFP cells, sample size, etc.

3. Line 91: The text states that “GFP+ embryos (identified by GFP excitation flashlight while embryos are in amnion) were carefully dissected from their sacrificial mother to avoid cross contamination of fetal and maternal cells.” As I read the rest of the study it refers to the study of the GFP- embryos for the presence of GFP+ cells. Is this a mistake or were the GFP+ embryos removed to avoid contamination of the GFP- embryos? If this is correct, perhaps this could be clarified by including in the schematic of Figure 1C.

4. Throughout the manuscript there is a continuous assumption that the source of the GFP+ cells is the mother, however there are other hypotheses described in the literature that could explain the source of microchimerism in utero. Assuming 50% of embryos in the litter are heterozygous (GFP+/-) it is difficult to determine the precise source of the cells. Anastomoses of placental vasculature has been previously demonstrated to produce twin-twin transfusion and could be another potential source of allogenic cells in utero. Were there any other measures used to confirm that the cells are of maternal origin? Is there any information or analyses that can be included about the heterozygous GFP+/- prevalence of each litter?

5. Lines 214-217: Could you elaborate on the possible causes of the seemingly low number of GFP+ cells measured in the embryos? This could perhaps be added as part of a broader discussion of limitations.

6. Lines 249-255: It is stated that the large proportion of GFP cells identified in one sample is a product of the mother, but it should be considered that this may be a result of tetragametic chimerism (the fusion of two embryos in utero) or possible transfusion from another embryo.

Minor comments

1. Lines 17-19: In the abstract it is stated that “maternal cells…migrate into the fetus and persist for the rest of their lives”, this appears to be an overstatement. While possible, there does not seem to be sufficient evidence to support this claim. It may be best to rephrase this into a phrase similar to “may persist for several decades” (see line 42).

2. Line 127: It is not clear if then entire material from each embryo was loaded onto the FACS machine or only a portion.

3. In lines 246-249 it is stated that this study identified a comparable number of MMc cells of previous studies reviewed by Kinder et al. However, on review the review article by Kinder et al. does not appear to summarize maternal microchimerism levels (although it does for fetal microchimerism). In this case it would be better to cite the specific studies that produced comparable results.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-14310R1Whole embryonic detection of maternal microchimeric cells highlights significant differences in their numbers among individualsPLOS ONE

Dear Dr. Naoki Irie,

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.

We thank you for your careful consideration of the reviewers' requests and suggestions, which has greatly improved the manuscript. However, a few clarifications and extra details have been asked by the reviewers, including discussing the influence of embryonic stage on the number of maternal cells.Please submit your revised manuscript by Nov 27 2021 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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We look forward to receiving your revised manuscript.

Kind regards,

Colette Kanellopoulos-Langevin, Ph.D

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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Comments to the Author

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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: N/A

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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

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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: please find details in the attached file

The authors have carefully taken into account the comments of the two reviewers.

Table 1 was absolutely essential and allows a substantiated argumentation of data as to the probable maternal origin of these cells.

Moreover this table allows to note that when embryonic stages increase, “maternal” Mc is more abundant. This could be one of the reason why the embryo for which the number of "maternal" cells was the highest was the one tested at the latest embryonic stage.

Thus, although it is undeniable that there is an heterogeneity between individuals for the number of maternal microchimeric cells, since the embryos do not have the same number of Mc cells for the same embryonic stage; it seems there is also an effect due to the embryonic stage.

Indeed a Mann Whitney test allows to see that the number of maternal cells is statistically higher at E16.5 (p=0.0147) compared to all other previous embryonic stages (E12.5 + E13.5 +E14.5 + E15.5).

Thus it is very possible that later embryonic stages are those where MMc is the most perfused. Nevertheless a Spearman correlation does not show a statistical increase with embryonic stages (p=0.3), only a tendency.

The fact that there appears to be an effect due to the embryonic stage needs to be mentioned at least in the discussion and probably in the results.

Reviewer #2: The manuscript “Whole embryonic detection of maternal microchimeric cells highlights significant differences in their numbers among individuals” details a technique for whole embryo analysis of maternal microchimerism. In a murine model the authors were able to utilize a breeding strategy of female mice heterozygous for fluorescent GFP with wild-type males to produce GFP- offspring. Using fluorescence-activated cell sorting, the authors were able to identify the presence of live GFP+ cells in some GFP- embryos, presumably originating from the mother. The study in several embryos found differences in GFP presence and quantity with one embryo presenting with a significantly larger proportion of GFP+ cells. The technique presented is well described and provides a useful approach for investigating the phenomenon of maternal microchimerism. The authors have appropriately addressed the reviewers’ comments and I am satisfied with the revisions made to address these concerns. In addition, I have a few additional comments that the authors should consider for the manuscript:

1. The primary objective stated in line 67 indicates testing the frequency of maternal microchimerism among individual embryos during normal pregnancy. The authors make the conclusion that there is similarity in the quantity of GFP+ cells between the embryos however there appears to be notable variation in qualitative GFP+ cell detection. Now seeing the detail and distribution of results provided in Table 1, there should be some additional discussion regarding variation between litters. For example, litter D and litter H with 100% prevalence of GFP+ cell detection compared to other litters C and I at the same gestational ages.

2. In the discussion section, lines 254-255, it is mentioned that “most of the embryos showed a comparable number of MMc cells, around 8 cells/ 10^7 sorted cells”. However, this estimation contradicts the data presented in Table 1 which (excluding the outlier) average 3.7 cells/10^7 sorted cells among samples with detectable GFP+ cells. Also, to improve clarity it would be helpful to indicate discussion of microchimerism results relating to “all embryos” or “GFP+ cell detected embryos”.

3. Further, in line 256-258 it is described that “one of the embryos at the latest stage (E18.5) showed 1,816 cells/10^7 cells, which corresponds to a frequency 1,000 times higher than that in the detected embryos”. This is true for some embryos with the lowest 1 cell/10^7 cells, but based on the average this would be approximately 500 times higher. Additional detail should be included in this sentence to help clarify the conclusions. This is also mentioned in the abstract line 27, and introduction line 71.

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Reviewer #1: Yes: Nathalie C. Lambert

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Review Plos One september 27-21.docx

Whole embryonic detection of maternal microchimeric cells highlights significant differences in their numbers among individuals

PONE-D-21-14310R2

Dear Dr. Naoki Irie,

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.

One minor requirement has been made by one reviewer regarding the most recent revision, and I agree with his suggestion:

 1. Lines 283-285: The added sentence regarding my previous comment on examining the ratio of GFP+ and GFP- embryos does not read clearly and may need to be revised. I believe the statement in parentheses is trying to say Litter C vs D and Litter H vs I show significant variation in the litter chimerism ratio at the same gestational age. Perhaps more concisely, “the litters at stages E13.5 and E16.5 show significant variation in the proportion of MMc+ detected embryos”.

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Colette Kanellopoulos-Langevin, Ph.D

Academic Editor

PLOS ONE

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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: 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

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.

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: All comments have been adequately addressed by the authors in this second revision. In particular

the fact that it is very possible that later embryonic stages are those where MMc is the most perfused, although statistical analyses show only a trend toward a correlation.

Reviewer #2: The authors of the manuscript “Whole embryonic detection of maternal microchimeric cells highlights significant differences in their numbers among individuals” describe their research using a technique to examine whole embryo microchimerism in murine models. This technique provides a novel insight into early stage transplacental maternal microchimerism and additional discoveries of variation in microchimerism presentation with the occasional incidence of significantly large cell populations. The authors have addressed the comments from both reviewers and made appropriate revisions to the manuscript. The work has shown significant improvement with the additional data and analyses that the authors have subsequently integrated into the final manuscript. I only have a minor comment regarding the most recent revisions.

1. Lines 283-285: The added sentence regarding my previous comment on examining the ratio of GFP+ and GFP- embryos does not read clearly and may need to be revised. I believe the statement in parentheses is trying to say Litter C vs D and Litter H vs I show significant variation in the litter chimerism ratio at the same gestational age. Perhaps more concisely, “the litters at stages E13.5 and E16.5 show significant variation in the proportion of MMc+ detected embryos”.

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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

Reviewer #2: Yes: Brandon N. Johnson