PONE-D-23-21148Topological defects in self-assembled patterns of mesenchymal stromal cells in vitro are predictive attributes of condensation and chondrogenesisPLOS 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: No

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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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 presented an in vitro method to predict the chondrogenic potency of MSCs. The authors examed the self-assembled patterns of confluent BM-MSCs and correlated these patterns with cartilage ECM productions. In general, the authors described a novel and easy method that predicts the chondrogenesis of MSC. However, there are some issues the authors should address.

The results section consists of a mixture of presenting and discussing the results. Please only present the results in the result section.

Line 166 - 168, quantitative data are required to support the claim.

Line 171 - 209, Figure S5 quantifies the number of clusters using nuclei count. Figure 5 quantifies the VoC of different patterns. What is the difference between the number of clusters and VoC? Is there a specific reason why the author changes the quantification method for Figure S5 and figure 5?

Line 192 - 193. Please present merged nuclei and Actin fluorescence images of the whole well to support the claim of "occurrence of condensation only for the topological defect region".

Line 256 - 257, the results of Fig S8 should be presented in the result section, not in the discussion section.

Line 267 - 268, the sex and age of the donors should be described.

In the method section, the authors should describe how they define different patterns: ordered, disordered, topological defects, and +1/2, +1 defects.

Reviewer #2: In this manuscript, the Authors have set up an algorithm for computational analysis of cell expansion in monolayer cultures. Despite the great novelty, several issues should be addressed.

1. Monolayer cultures of MSCs can be used for chondrogenesis; however, other methods have been widely described and adopted, such as spheroids and 3D high-density cultures, with a more efficient chondrogenesis (e.g., Lamparelli EP, Ciardulli MC, Giudice V, Scala P, Vitolo R, Dale TP, Selleri C, Forsyth NR, Maffulli N, Della Porta G. 3D in-vitro cultures of human bone marrow and Wharton's jelly derived mesenchymal stromal cells show high chondrogenic potential. Front Bioeng Biotechnol. 2022 Sep 26;10:986310. doi: 10.3389/fbioe.2022.986310).

2. It is not well discussed how confluency in monolayer culture can be linked to effective chondrogenesis, or how actin expression and orientation can be a good marker of effective chondrogenesis, while results from chondrogenic-related proteins has been added only in supplementary files without any discussion. Moreover, chondrogenesis is triggered by appropriate stimuli in culture medium and requires several days (at least 15 days), while staining and cultures end at day 10 or 12.

3. No data on effective chondrogenesis are reported, and no data on exclusion of different lineage differentiation or fibrotic shift are shown.

4. In vitro chondrogenic differentiation assay conditions are different from those used for setting up the method.

5. A negative control without chondrogenic medium or with a different differentiation medium is not described.

6. Order of references should be corrected and abbreviations should be always used once defined.

7. Major English check should be performed.

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Reviewer #1: No

Reviewer #2: No

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Topological defects in self-assembled patterns of mesenchymal stromal cells in vitro are predictive attributes of condensation and chondrogenesis

PONE-D-23-21148R1

Dear Dr. Van Vliet,

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,

Nileshkumar Dubey

Academic Editor

PLOS ONE

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