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The authors addressed all the reviewers concerns. The publication is now ready for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
We have carefully reviewed your work and R2 has identified some minor issues that need to be addressed. Please ensure each of the raised issues is thoroughly addressed
The author has diligently attended to all comment suggestions and corrections, effectively addressing each one with thorough responses and revisions
The author has diligently attended to all comment suggestions and corrections, effectively addressing each one with thorough responses and revisions
The author has diligently attended to all comment suggestions and corrections, effectively addressing each one with thorough responses and revisions
The author has diligently attended to all comment suggestions and corrections, effectively addressing each one with thorough responses and revisions
The manuscript has indeed improved with a better flow of ideas. Just a small typo on line 369 - UCMSCP3N1 is repeated twice.
No comment
The authors answered the questions nicely. While it’s always nice to see well separated clusters, I understand that this pattern might occur in cells with similar phenotype.
However, as for previous comment 9 (for the statistical analyses), the supporting tables 2 and 3 have only the proportional composition in %. There is no statistical analysis on this. I think that a statistical test such a Chi-squared goodness of fit test would improve the authors’ claims. This test is used to evaluated proportions of categorical or discrete data, which I think could be adequate for this type of data.
Also, decimal places in the tables can be shortened to two to make it easier to read.
The quality of the article has greatly improved since last submission. The authors answered nicely to all the comments.
While more experiments are necessary to conclude that a specific population or culture condition has more effect on a specific path, the data does not contradict what the authors aimed to study and their conclusions.
Therefore, I think that the article is suitable for publication.
We have carefully reviewed your work and appreciate the insights it offers to the field. While your submission has great potential, the reviewers have raised some valid concerns that need to be addressed for the manuscript to reach its full potential. Kindly take the time to thoroughly address each of the raised issues, providing clear justifications.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
**Language Note:** PeerJ staff have identified that the English language needs to be improved. When you prepare your next revision, please either (i) have a colleague who is proficient in English and familiar with the subject matter review your manuscript, or (ii) contact a professional editing service to review your manuscript. PeerJ can provide language editing services - you can contact us at [email protected] for pricing (be sure to provide your manuscript number and title). – PeerJ Staff
Keywords:
Line 49: Suggestion – change mesenchymal stem cells to mesenchymal stromal cells to expand discoverability. ‘Mesenchymal stem cells’ is already mentioned in the title.
Introduction:
Line 57: To put space before ‘However..’
Line 79: Suggestion - Add a sentence or 2 to connect between paragraph 2 and 3 of the introductions, from the context of MSC heterogeneity to specialized differentiation potential of MSC in angiogenesis and reproductive system.
Line 91: Add reference for this statement - ‘Additionally, MSCs exhibit immunomodulatory properties, suppressing autoimmune responses in reproductive diseases.’
Supplement materials:
Supplementary Figure 2. Identification of CMMSCs add on meaningful labels to indicate positive induction for F, G and H.
Introduction:
Line 97-98: Comment on the basis of selection of all the four types of tissue sources. What would be the importance of characterising these types of MSC sources compared to other more therapeutically accepted stem cell source for instance MSC from the bone marrow?
Method:
Line 143-145: Please recheck this protocol for missing step – ‘Umbilical cord: The umbilical cord tissue was rinsed with DPBS (Gobio, 14190250), then cut into 144 1.5 cm fragments, and had blood vessels and outer membranes removed. The processed tissue was then digested with an equal volume of 0.2% type I collagenase (Sigma, C0130) for 40 minutes.’ to ensure clarity and reproducibility of the procedure.
Line 153: ‘…type 1 collagenase..’ is usually written as type I (roman).
Line 160-161: ‘…and hypoxic conditions were maintained at 1% oxygen concentration.’ – state the protocol to establish hypoxic condition for this research (add reference if necessary) and clarify the indicator that the hypoxic condition was successfully achieved in this model.
Line 170: State MSCs at which passage were used for all the lineage differentiation in vitro.
Line 173: Correct ‘CO2’ CO2 and remove space after comma.
Line 175: To put space before ‘To confirm…’
Line 189: 2-3 biological replicates were used for single cell RNA sequencing. Comment on this small sample size and justify the appropriateness of data obtained in representing the true signature/profile of the investigated cell source.
Results:
Line 264: Clarify number of donors, mentioned 10 in method section but 9 in result section.
Line 264: Remove space after (Supplementary Table 1)
Line 343: Correct ‘…whether alterations in vitro culture conditions…’
Line 345: Correct spacing ‘(Table S1) ,spanning…‘
Line 420: Correct spacing
Discussions:
Line 278-281 and Line 312-314: ‘This observation suggests that there may be meaningful differences in the number and composition of subpopulations of MSCs among different tissue sources, which is crucial for understanding the diversity and potential applications of MSCs.’ And ‘Notably, the C5 subpopulation constitutes over 80% of CMMSCs but is
less prevalent in AMMSCs and UCMSCs. Most strikingly, this subpopulation is nearly absent in ADMSCs.’– It would be interesting to read some discussions for these findings.
Provide discussion/comment on the possible association of specialized functionality found in each source of MSCs to its tissue origin and structure.
This research explores the diverse characteristics of mesenchymal stem cells (MSCs) and how they are influenced by tissue source, passage number, and culture conditions, crucial for regenerative medicine. Utilizing single-cell transcriptome profiling, MSCs from adipose tissue, chorionic villi, amniotic membrane, and umbilical cord, including different passages and low-oxygen conditions, were analyzed. Results reveal distinct functional traits among MSCs from different tissues, such as adipose tissue MSCs showing potential for vascular regeneration and chorionic villi MSCs associated with reproductive processes. Changes in subpopulation proportions were noted with varying passages and culture conditions, particularly in vascular and reproductive system regeneration contexts. This research offers insights into optimizing personalized regenerative medicine treatments, especially for conditions involving vascular and reproductive systems.
Ning Yi and colleagues describe here the transcriptome profile of mesenchymal stem cells from different sources, as well as distinct culture conditions. The field background is sufficient with appropriate context, with the literature gap identified and addressed.
However, some English improvements need to be performed throughout the text. For instance, line 291-292, the phrase starts with “although” but then it ends abruptly. Also, “etc” should be avoided in scientific writing and replaced with the specific words. Also, despite the abbreviations being stated in the abstract they should be repeated in the main text.
The figures are overall well-structured, but the supplementary ones lack resolution with the scale bar not perceptible in some. Also, the subtitles should be corrected as it states the wrong letter (F,G and H, sometimes are referred as A, B and C. Moreover, in the methods it it’s that alizarin red s is used but, in the image subtitle says 0.2% Xylene Red.
Please correct this for improved clarification.
The primary research is within the aims and scope of the journal, with the research question well defined and addressed. However, some major concerns rose that may affect the paper quality.
Methods are confusing regarding samples. Line 264 it states 9 healthy donors, while the supplementary table 1 describe 10 donors for that analysis. Also, it’s not well described what the parameter is referring to. It ranges from 2250 g to 72 kg, which implies is different types of measurements.
Also some text in the methods should be in another sections. For example, line 122-135.
Moreover, do the isolations followed established protocols? No reference is provided.
More than passage, population doublings would be more appropriate, as different cells could be in the same passage but with different population doublings. Also, it should be addressed why in the first only passage 5 was analyzed.
The authors should also explain why different exclusion parameters were used for different parameters (Line 216 -222)
And also why only umbilical cord MSCs were used for the study of hypoxia and passage number. And why hypoxia was only performed on passage 5.
Despite a good approach to fill the gap in the literature, some concerns rose when reviewing the article that might affect the validity of the findings.
In the first part of the manuscript, the authors identify 6 clusters in the data (C0-C5). However, upon visualization of UMAPs, the clusters do not seem properly separated, with big overlapping between the clusters, which may indicate that different clustering parameters should be assessed as well as other dimensionality reduction techniques. This also happens in the second part of the work. Without defined clusters, the conclusions that come after are severely affected.
In line 371-373, the authors state that there are no significantly distinct markers genes among these subpopulations, which may indicate that the cells heterogeneity is not defined by the cluster classification.
Moreover, comparisons between groups on figure 2 and 4 are only done in a visual manner, without any statistical analysis to back some claims. For example, on line 352, the authors state there were relatively minor differences among groups in figure 4A. However, some do not seem minor as the low oxygen group do not present any S5 cluster and presents an increase in the S4 cluster. Major and minor differences are relative terms and more defined statistical comparison is recommended to strengthen the claims.
Regarding the low oxygen study, according to the supplementary table, the cells that were grown on low oxygen conditions are from different donors than the cells grown on normal oxygen conditions. Therefore, it is dubious if the phenotype observed is from donor heterogeneity or from hypoxia conditioning. This type of analysis should be performed on the same donor to make direct comparisons.
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