The Effects of Co-Culture of Embryonic Stem Cells with Neural Stem Cells on Differentiation
Round 1
Reviewer 1 Report
The Article is devoted to an interesting question, if there is a difference between the effects of adjacent cells or cellular products on stem cell differentiation, as finding the technology of in vitro embryonic stem cells differentiation into neural lineages attracts scientists’ attention in fundamental and practical aspects.
The research is an elegant, well planned and illustrates the peculiarities of the different types of impacts, including chemical stimuli only or both chemical and physical stimuli, on embryonic stem cells differentiation in 2D or 3D cultures.
The study is logically structured and the performed experiments serve perfectly the goal to answer the main question which Authors formulated.
The methods that have been used during the research are quite sensitive and had been selected with a great accuracy. The interesting approach of 3D spheroids formation had been chosen using plates with micro-wells in the form of concave, which allows to gently get the equal cellular aggregates without any additional impact towards the cells.
The results are described in details. The obtained data help to understand the peculiarities of conditioned medium and co-culture approach influence on embryonic stem cells differentiation in 2D and 3D cultures, which is very important as it gives an opportunity to reveal the specificity of the cellular environment effect on stem cells fate.
The following comments do not diminish the value of the Article:
Line 61 Probably it would be better to use abbreviation MSCs?
Line 97 Probably it has to be 1,000 U/mL?
Line 121, Line 140 ‘35π dishes’ is that for 35 mm dish?
Line 134 ‘60π dishes’ is that for 60 mm dish?
Probably it also would be quite informative to add to Methods section the description of statistical methods that had been used during the data processing.
Line 195 It would be interesting to have a bit more detailed explanation of flow cytometry analysis of the percentage of Oct4-GFP-negative cells cultured on NSC or MEF feeders (the data shown on the Figure 1). Would it be possible to add an information about the scale size located on ‘b’ part of the Figure 1?
Line 220 Would it be possible to enlarge a bit an information about the scale size located on ‘b’ part of the Figure 2, so it would become visible?
Line 228 Would it be possible to enlarge a bit an information about the scale size located on ‘b’ part of the Figure 3?
Line 234 The meaning of the lowercase letters which represent significant differences in the expression levels in various mediums is not very much clear, does it mean that every parameter differs from all others? Might the meaning of the lowercase letters be specified a bit more?
Line 248 It would be better to increase a bit font size of the text on the ‘c’ part of the Figure 4.
Line 248 Would it be possible to enlarge a bit an information about the scale size located on ‘b’ part of the Figure 4?
It would be very interesting to know the content of CM.
The Conclusions section should be added as it is recommended on the website of the Journal.
Author Response
Response to Reviewer 1 Comments
Response: We would like to thank the reviewer for positive feedback regarding our manuscript and work!
Point 1: Line 61 Probably it would be better to use abbreviation MSCs?
Response 1: Thanks for the comments. According to this suggestion, we have modified the sentence.
Point 2: Line 97 Probably it has to be 1,000 U/mL?
Response 2: Thanks for the comments. I tried to write it as 103, but it seems to be a typo. According to this suggestion, we have modified the sentence.
Point 3: Line 121, Line 140 ‘35π dishes’ is that for 35 mm dish?
Response 3: Thanks for the comments. According to this suggestion, we have modified the sentence.
Point 4: Line 134 ‘60π dishes’ is that for 60 mm dish?
Response 4: Thanks for the comments. According to this suggestion, we have modified the sentence.
Point 5:. Probably it also would be quite informative to add to Methods section the description of statistical methods that had been used during the data processing.
Response 5: Thanks for the comments. According to this suggestion, we have added the description of statistical methods that had been used during the data processing to Methods section.
Point 6:. Line 195 It would be interesting to have a bit more detailed explanation of flow cytometry analysis of the percentage of Oct4-GFP-negative cells cultured on NSC or MEF feeders (the data shown on the Figure 1). Would it be possible to add an information about the scale size located on ‘b’ part of the Figure 1?
Response 6: Thanks for the comments. According to this suggestion, we have added a bit more detailed explanation of flow cytometry analysis of the percentage of Oct4-GFP negative cells cultured on NSC or MEF feeders (the data shown on the Figure 1). Also, we have added an information about the scale size located on ‘b’ part of the Figure 1.
Point 7: Line 220 Would it be possible to enlarge a bit an information about the scale size located on ‘b’ part of the Figure 2, so it would become visible?
Response 7: Thanks for the comments. We have modified the scale size located on ‘b’ part of the Figure 2.
Point 8: Line 228 Would it be possible to enlarge a bit an information about the scale size located on ‘b’ part of the Figure 3?
Response 8: Thanks for the comments. We have modified the scale size located on ‘b’ part of the Figure 3.
Point 9: Line 234 The meaning of the lowercase letters which represent significant differences in the expression levels in various mediums is not very much clear, does it mean that every parameter differs from all others? Might the meaning of the lowercase letters be specified a bit more?
Response 9: Thanks for the comments. We have modified the Figure legend of the Figure 3 in Result section.
Point 10: Line 248 It would be better to increase a bit font size of the text on the ‘c’ part of the Figure 4.
Response 10: Thanks for the comments. We have modified the font size of text on the ‘c’ part of the Figure 4.
Point 11: Line 248 Would it be possible to enlarge a bit an information about the scale size located on ‘b’ part of the Figure 4?
Response 11: Thanks for the comments. We have modified the scale size located on ‘b’ part of the Figure 4.
Point 12: It would be very interesting to know the content of CM.
Response 12: Thanks for the comments. The CMs were made by using FBS, FBS+LIF and NSC medium on MMC treated NSC or MEF. The detail content of FBS, FBS+LIF and NSC medium was mentioned in “2.1 Types of culture medium” of manuscript.
Point 13: The Conclusions section should be added as it is recommended on the website of the Journal.
Response 13: Thanks for the suggestion! According to this suggestion, we have inserted a paragraph of “Conclusions” to underline the relevance of the obtained results.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The authors (Ye Rim Kim and colleagues) presented their manuscript where they evaluated “The effects of co-culture of embryonic stem cells (ESC) with neural stem cells (NSC) on differentiation”. In detail, the authors used two- & three-dimensional culture conditions and NSC-derived conditioned medium for ESC differentiation. They concluded that co-culture with NSC elevates ESC differentiation into ectoderm. Although the manuscript generally presented in a logical order, the text should be revised to improve clarity of the writing and I believe after substantial input from the authors in this manuscript may considered for publication in Curr. Issues Mol. Biol., MDPI. The author should consider the following minor issues to improve the strength of this manuscript:
Comments:
• Definition of co-culture is that “a cell culture containing two or more different types of cells.” All over the manuscript authors define it “more than two” but it would be “two or more”. Correct it.
• Mention the full form of first time used abbreviate term. Especially in the introduction, authors used abbreviate term of the targets (such as, - Flk-1, PDGFR, BMP, Wnt, etc.) without mentioning the full form.
Also, the term conditioned medium (CM), as the authors already mentioned the full form of it in the abstract either don’t define it again in other section of the manuscript or mention the same for it when the term is appearing first time in each section such as abstract, introduction, results, etc.
• In section 2.10 Equipment, minimum description needed. Which pictures, in how much magnification, what color, etc.
• In line 184, “FBS, without LIF,”. I believe, it should be “FBS with or without LIF,” Correct it.
• It’s difficult for readers to understand the results if the figures won’t be arranged such a way that they can be describe in the result section sequentially (like A, B, C, …....). For example, - in the section “3.1 NSC feeder induced differentiation of ESCs”, authors first describe Figure 1A then 1C (line 182) then Figure 1B and again back to Figure 1C. Authors need to be more careful when they will arrange the figures to describe in the result section.
Also, the authors used small letters in the figures but capital letters in the result section for numbering.
Author Response
Response to Reviewer 2 Comments
Response: We would like to thank the reviewer for positive feedback regarding our manuscript and work!
Point 1: Definition of co-culture is that “a cell culture containing two or more different types of cells.” All over the manuscript authors define it “more than two” but it would be “two or more”. Correct it.
Response 1: Thanks for the comments. We have deleted entire sentence as necessary.
Point 2: Mention the full form of first time used abbreviate term. Especially in the introduction, authors used abbreviate term of the targets (such as, - Flk-1, PDGFR, BMP, Wnt, etc.) without mentioning the full form. Also, the term conditioned medium (CM), as the authors already mentioned the full form of it in the abstract either don’t define it again in other section of the manuscript or mention the same for it when the term is appearing first time in each section such as abstract, introduction, results, etc.
Response 2: Thanks for the comments. According to this suggestion, we have modified the sentences and added information for abbreviate term of the targets (such as, Flk-1, PDGFR, BMP, Wnt, etc.). Also, we have revised the term conditioned medium to CM in each section.
Point 3: In section 2.10 Equipment, minimum description needed. Which pictures, in how much magnification, what color, etc.
Response 3: Thanks for the comments. We have added the additional information about 2.10 Equipment section.
Point 4: In line 184, “FBS, without LIF,”. I believe, it should be “FBS with or without LIF,” Correct it.
Response 4: Thanks for the comments. According to this suggestion, we have modified the sentence.
Point 5:. It’s difficult for readers to understand the results if the figures won’t be arranged such a way that they can be describe in the result section sequentially (like A, B, C). For example, - in the section “3.1 NSC feeder induced differentiation of ESCs”, authors first describe Figure 1A then 1C (line 182) then Figure 1B and again back to Figure 1C. Authors need to be more careful when they will arrange the figures to describe in the result section.
Also, the authors used small letters in the figures but capital letters in the result section for numbering.
Response 5: Thanks for the comments. According to this suggestion, we have modified the numbering in the result section. Also, we have rearranged the figures 1b and 1c in the result section.
Author Response File: Author Response.docx
Reviewer 3 Report
The Manuscript by Ye Rim Kim and colleagues entitled “The effects of co-culture of embryonic stem cells with neural 2 stem cells on differentiation” deals with two- and three-dimensional co-cultures of embryonic stem cells (ESCs) with neural stem cells (NSCs) which contribute to the differentiation of ESCs into the ectoderm.
The following sentence in the Abstract “A co-culture is a culture system that constitutes more than two different types of cells in a culture dish” is needless.
Acronyms for all factors mentioned should be included, e.g. FGF, Wnt, BMP.
Please insert the scale bar of the images in all figures, either those in phase contrast or in fluorescence.
I would suggest the Authors to insert a paragraph of "Conclusions" to underline the relevance of the obtained results.
I would ask the authors to insert a schematic figure to summarize the most important aspects of the work.
Author Response
Response to Reviewer 3 Comments
Response: We would like to thank the reviewer for positive feedback regarding our manuscript and work!
Point 1: The following sentence in the Abstract “A co-culture is a culture system that constitutes more than two different types of cells in a culture dish” is needless.
Response 1: Thanks for the comments. According to this suggestion, we have deleted the following sentence in the Abstract “A co-culture is a culture system that constitutes more than two different types of cells in a culture dish” and modified the next sentence to fit the context.
Point 2: Acronyms for all factors mentioned should be included, e.g. FGF, Wnt, BMP.
Response 2: Thanks for the comments. According to this suggestion, we have added information for abbreviate term of the targets (such as, FGF, BMP, Wnt, etc.).
- Please insert the scale bar of the images in all figures, either those in phase contrast or in fluorescence.
Response 3: Thanks for the comments. We have inserted the scale bar of the images in all figures, either those in phase contrast or in fluorescence. - I would suggest the Authors to insert a paragraph of "Conclusions" to underline the relevance of the obtained results.
Response 4: Thanks for the suggestion! According to this suggestion, we have inserted a paragraph of “Conclusions” to underline the relevance of the obtained results. - I would ask the authors to insert a schematic figure to summarize the most important aspects of the work.
Response 5: Thanks for the comments. We have inserted a schematic figure to summarize the most important aspect of the work.
Author Response File: Author Response.docx
