Role and Function of Mesenchymal Stem Cells on Fibroblast in Cutaneous Wound Healing

Round 1

Reviewer 1 Report

Please see the attachment.

Comments for author File: Comments.docx

Author Response

Thank you for your valuable comments. We revised the manuscript as you pointed out.

Tanaka and collaborators aimed to digest current knowledge on the role of mesenchymal stem cells in the healing of wounded skin. This is an important process, in which multiple MSC activities may be recruited. The interest of this review article has been, however, compromised by multiple flaws.

It is not always clear whether the authors are referring to conventional MSCs grown in culture as therapeutic cells, or to “natural” MSCs present in tissues. It seems that the authors consider that bona fide MSCs are present in the organism, and are established in culture for sustained proliferation. In fact, MSCs have been isolated, identified, and characterized in vitro, and hence are by definition cultured cells. MSCs are a culture phenomenon. Whether a cell identical to the cultured, conventional MSC exists in vivo is not known. Some cell types, in particular perivascular cells, have been described as MSC native ancestors, but this has not been discussed at all in the review. Hence, the assumption that tissue-resident MSCs are involved in wound healing should be put forward with prudence and discussed in more depth.

Thank you for your suggestions. We added future prospect of MSCs focusing on cutaneous wound healing.

3.6. Future prospects of MSCs

Currently, research institutes and companies are developing methods for the mass culture of high-quality MSCs, but there are very few successful examples and cannot be applied in clinical settings. Therefore, researchers and physicians are carefully culturing in flasks one by one and administering them to individual patients. The establishment of a simple and accurate mass culture method for MSCs is one of the issues that must be solved immediately. Even when used for wound healing, MSCs will be able to be administered via intravenous infusion or topical injection without culture simultaneously [130]. A difficult administration method will cause confusion in the clinical setting, and itself makes difficult to further development. Ideally, MSCs should be administered in a manner that promotes rapid wound repair, similar to the administration of antimicrobial agents. Furthermore, it is hoped that the exosomes and wound-healing substances secreted by MSCs could be used intravenous injection instead of using MSCs in itself, and these substances could provide to patients more conveniently due to development of self-administration drugs.

With respect to the title of the article, many sections are off-topic. For instance, the long discussion on the respective properties of bone marrow and fat derived MSCs is not relevant here. The same applies to the discussion on the bone marrow hematopoietic stem cell niche, and associated references, which have nothing to do in this review. In fact, I found less than 30 references directly relevant to skin wound healing, out of 116 cited. Many references do appear anecdotal. Conversely, landmark articles have not been cited, such as Crisan, Cell Stem Cell 2008, or Fiona Watt’s studies on skin fibrosis. Lack of focus and absence of some key references are major weaknesses of this article.

We really thank you for your valuable comments. We added about 20 references as you pointed out.

The review is disjointed; individual paragraphs should be better organized: we often find a succession of anecdotal findings, with no real logical progression. Surface markers of MSCs, factor secretion by MSCs… such topics are mentioned randomly in different sections of the article and should rather be evoked synthetically and logically in devoted sections. There are also multiple repeats: for instance, how many times are we told that fat derived MSCs are preferred to BM derived counterparts?... Making the point once should be enough. 

While some sections are far too long (see above), the conclusion is much too short

Thank you for your suggestions. As you pointed out, we summarized the indicated section and expanded the conclusions.

4. Conclusion

Accelerated wound healing and proper collagen production by fibroblasts is a very important process as it prevents scar formation. MSCs have long been used in regenerative medicine owing to fewer ethical issues, relatively easy to harvest from organisms, small risk of immune rejection if self-derived MSCs are used, smaller risk of tumor formation compared to induced pluripotent stem cells and ES cells. In recent years, MSC-derived exosomes have attracted attention for their inflammation-modulating properties, and their application in wound healing is expected. Since MSCs and fibroblasts are closely interrelated, fundamental elucidation of these mechanisms will greatly contribute to regenerative medicine especially in plastic surgery and dermatology. However, some reports are skeptical about whether MSCs act on fibroblasts in the skin or affect wound healing [130-133]. The significance of using stem cells for chronic wound healing has been discussed, and there is a growing understanding of the mechanisms and pathophysiology.

                                                                                                                                                                                                                     

Other comments and examples (with reference to line numbers). The list is not exhaustive:

20 “MSCs are pluripotent stem cells”. MSCs are not pluripotent cells

We rewrote “MSCs are multipotent stem cells.” (line 20)

24 “Moreover, MSCs exhibit anti-inflammatory and anti-fibrotic effects via paracrine pro-inflammatory cytokines”??

We rewrote “via paracrine signalling.” (line 25)

32 “stem cells of biological origin” Is any stem cell of non-biologic origin?

We delete: of biological origin (line 31)

36 “As MSCs are harvested and cultured from living organisms, there are few ethical issues around their use” This sentence is impossible to understand. Stem cells from dead organisms?.. What does this have to do with ethics? Please explain

Thank you for your comments. In general, MSCs can be harvested from the patient and injected to the patient simultaneously (Akita S, et al. Clin Plast 2012). Moreover, we do not need culture the MSCs because of rich sources. So, we changed the sentences. “On the other hand, embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre-implantation embryo. ES cells for clinical use have always raise ethical issues. However, researchers and physicians can avoid ethical problems in MSCs transplantation because MSCs can be harvested and cultured from adult tissues”. (line 35–39)

 

39 “Fibroblasts make up all tissues in the body” What is the meaning of this? Please explain

We changed make up to”consist”. (line 41)

54 “MSCs are harvested and isolated from bone marrow and adipose tissue using a flow cytometer”. This is inexact. Pre-MSCs are only isolated by FACS for research purposes. In the vast majority of cases, MSCs are grown in primary cultures of unselected cells

We wanted to explain method to collect pure MSCs. So, we rewrote the Figure legends. Collecting pure MSCs is essential to apply MSCs to clinical medicine. That is why we made minor corrections in Figure1.  (line 54–59)

105 “Clinical trials with MSCs already exist” This is an understatement, with over 1200 trials to date. The first trials took place in the the early 1990’s

Thank you for your valuable comments. We rewrote this sentence using Reference44 (Rodríguez-Fuentes DE, et al. Mesen-chymal Stem Cells Current Clinical Applications: A Systematic Review. Arch Med Res. 2021) “There are approximately 1138 clinical trials exists with MSCs by July 2020”. (line 106)

114 “seeding bone marrow mononuclear cells on culture dishes and collecting fibroblast-like colony-forming cells (CFU-F)” The CFU-f is the precursor. You do not collect CFUs-f but the progeny thereof, i.e. MSCs

We changed the sentence “The MSCs that are currently available are obtained by seeding bone marrow mononuclear cells on culture dishes and collecting attached cells after 2–3 weeks of culture.”

124 “MSCs lose their migration ability” Was it ever demonstrated that MSCs have migratory ability, to start with?

We added the reason and changed the sentence “When MSCs pass through the blood vessels of the lungs, they should be trapped in the small capillaries of the lungs. [61,62].” (line 124–126)

145 “defined as non-hematopoietic colony-forming cells” This could be written 20 years ago, not nowadays

As you pointed out, there are still controversial for the questionnaire.

We rewrote the sentence “MSCs are found in a variety of tissues in vivo and have been extensively studied, particularly the bone marrow-derived.” (lines 145–147)

 

157 “Furthermore, separation methods using antigen-antibody reactions have been studied using fluorescently labeled antibodies as markers for proteins on the cell surface of MSCs” Related statements have been made multiple times in this review. Please be more concise and synthetic

We deleted the sentence as you pointed out. Thank you.

189 “the number of MSCs that can ultimately be secured is also small”. This is not true. Large, clinically relevant numbers of MSCs can be collected from cultured BM (the average dose per patient is around 200 million cells)

Thank you for your suggestion. That sentence is not what we meant. We rewrote the sentence “the number of MSCs that retain multilineage differentiation potential is small”. (lines 187–188)

196 “The convenience of harvesting many MSCs simultaneously has led many researchers to clinically apply adipose-derived MSCs” Related statements have been made multiple times in this review. Please be more concise and synthetic

We deleted it. Thank you.

207 “Adipose tissue-derived MSCs are multipotent and can differentiate into numerous cell forms such as osteocytes, adipocytes, neural cells, vascular endothelial cells, myocytes, pancreatic ß cells, and hepatocytes” Who still believes that MSCs can differentiate into neural cells, endothelial cells, ß cells, hepatocytes? Please be more critical of published data

We added references (Nos. 80–84 81.                Moghadam FH, Alaie H, Karbalaie K, Tanhaei S, Nasr Esfahani MH, Baharvand H. Transplantation of primed or unprimed mouse embryonic stem cell-derived neural precursor cells improves cognitive function in Alzheimerian rats. Differentiation. 2009;78(2-3):59–68. doi: 10.1016/j.diff.2009.06.005.

82. Ikhapoh IA, Pelham CJ, Agrawal DK. Sry-type HMG box 18 contributes to the differentiation of bone marrow-derived mesenchymal stem cells to endothelial cells. Differentiation. 2015;89(3-4):87–96. doi: 10.1016/j.diff.2015.03.003.
83. Lee JH, Kosinski PA, Kemp DM. Contribution of human bone marrow stem cells to individual skeletal myotubes followed by myogenic gene activation. Exp Cell Res. 2005;307(1):174–82. doi: 10.1016/j.yexcr.2005.03.008.
84. Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Quinn G, Okochi H, Ochiya T. Adipose tissue-derived mes-enchymal stem cells as a source of human hepatocytes. Hepatology. 2007;46(1):219–28. doi: 10.1002/hep.21704.). (lines 204–207)

224 “Since the maintenance of MSC undifferentiability is largely unknown, we should compare bone marrow-derived MSCs and adipose tissue-derived MSCs in terms of MSC undifferentiability and stem cell properties in the future study” Please state what you mean more clearly

We rewrote the sentence “The mechanisms of maintenance of MSC multipotency is still unrevealed. We believe that the differences of MSC characteristics are affected by the differences of their origin. Further research is needed to investigate the characteristic of MSCs multipotency and apply for regenerative medicine.” (lines 222–224)

 

227 “We believe that further research on the mechanisms of stem cell immaturity is required” What is “stem cell immaturity”?.. Please explain

Thank you for your question. The sentence was shallow. We delete the sentence.

228 “Further elucidation of the undifferentiated nature of MSCs derived from adipose tissue has great potential for the development of regenerative medicine” This kind of very general statement is neither very clear nor useful. The authors should rather propose a strategy

We added the sentence “Specifically, MSCs derived from adipose tissue increased renal blood flow and improved renal function when administered to patients with focal seg-mental glomerulosclerosis [87]. The in vivo kinetics of the MSCs used in this clinical trial have not been elucidated, and the mechanism may be infestigated using gene modification techniques.” (lines 226–231)

 

233 “The mechanisms by which MSCs promote wound healing include secretion of angiogenesis-promoting factors, promotion of angiogenesis through differentiation into endothelial cells and pericytes” Pericytes have been actually proposed at the origin of MSCs, but this has been completely overlooked by the authors

As you pointed out, we deleted “and pericytes”. (line 237)

276 “When normal skin tissue is damaged by external factors, MSCs migrate to the damaged area via leaked blood” Are we considering here “natural” MSCs, or conventional cultured MSCs used therapeutically? Where is the experimental demonstration this is the way MSCs function? This reviewer is not aware of such a proof. This statement should be only made as a mere hypothesis

We added a reference [103] (Lynch MD, Watt FM. Fibroblast heterogeneity: implications for human disease. J Clin Invest. 2018;128(1):26–35. doi: 10.1172/JCI93555). Lynch et al. reported how fibroblast heterogeneity may provide insights into pathological states including wound healing (line 279)

 318 “breast milk”. Isn’t milk always from breast?

We rewrote it. (line 330)

321 “exosomes from bone marrow-derived MSCs have a high regenerative capacity, those from adipose tissue-derived MSCs have a high immunomodulatory capacity, and those from umbilical cord-derived MSCs have high tissue damage repair capacity” This refers to only one publication. The authors should be more prudent and reserved

We put one sentence “Although only one case has been reported.” (lines 332–333)

349 “MSCs have long been used in regenerative medicine owing to fewer ethical issues” The ethical aspect is only a marginal reason for using MSCs. Real benefits of MSCs are elsewhere

We rewrote the sentence “MSCs have long been used in regenerative medicine owing to fewer ethical issues, relatively easy to harvest from organisms, small risk of immune rejection if self-derived MSCs are used, smaller risk of tumor formation compared to induced pluripotent stem cells and ES cells.” (lines 377–379)

Reviewer 2 Report

• Figure 1 is too general. Add more details to the figure.
• Make a table and compare MSCs function on fibroblast in different studies under different conditions.
• Present the history of using MSCs to date as a schematic figure.
• The authors can add a “future prospects” section at the end of the manuscript.
• The authors can use the following reference in this study:

Sabbagh, F., & Kim, B. S. (2022). Recent advances in polymeric transdermal drug delivery systems. Journal of Controlled Release, 341, 132-146.

 

Author Response

Thank you for your valuable comments and suggestions. We revised the manuscript as you pointed out.

Figure 1 is too general. Add more details to the figure.

As you pointed out, we wanted to explain method to collect pure MSCs. So, we rewrote the Figure legends. Collecting pure MSCs is essential to apply MSCs to clinical medicine. That is why we made minor corrections in Figure1

 

Make a table and compare MSCs function on fibroblast in different studies under different conditions.

We added Table about MSC functions on fibroblast under different conditions (line 311–317)

 

Present the history of using MSCs to date as a schematic figure.

Unfortunately, the history of using MSCs is very complicated. That is why we cannot a schematic figure. However, instead of that, we added these sentences.

“MSCs were first isolated from  bone marrow in 1960-1970’s and were characterized as cells with fibroblastoid shape and osteogenic potential, which form clonogenic colonies [22] (line 98-100)

There are approximately 1138 clinical trials exists with MSCs by July 2020 [44]”.(line108)

The authors can add a “future prospects” section at the end of the manuscript.

We added 3.6. Future prospects of MSCs. (lines 359­–372)

3.6. Future prospects for MSCs

Currently, research institutes and companies are developing methods for the mass culture of high-quality MSCs, but there are very few successful examples and cannot be applied in clinical settings. Therefore, researchers and physicians are carefully culturing in flasks one by one and administering them to individual patients. The establishment of a simple and accurate mass culture method for MSCs is one of the issues that must be solved immediately. Even when used for wound healing, MSCs will be able to be administered via intravenous infusion or topical injection without culture simultaneously [130]. A difficult administration method will cause confusion in the clinical setting, and itself makes difficult to further development. Ideally, MSCs should be administered in a manner that promotes rapid wound repair, similar to the administration of antimicrobial agents. Furthermore, it is hoped that the exosomes and wound-healing substances secreted by MSCs could be used intravenous injection instead of using MSCs in itself, and these substances could provide to patients more conveniently due to development of self-administration drugs.

 

The authors can use the following reference in this study:

Sabbagh, F., & Kim, B. S. (2022). Recent advances in polymeric transdermal drug delivery systems. Journal of Controlled Release, 341, 132-146.

We added the reference in this manuscript.

Reviewer 3 Report

This is a very interesting review. about the interaction of MSCs and fibroblasts in wound healing. The paper is well written and every chapter is deeply thought and represented. However it's my opinion that is necessary to rewrite the chapter about the wound healing because in the decription of this one are completedy missed the contribution of some cells (for example mast cells). Besides about the interactions beetween the MSc and fibroblasts, is necessary to enlarge the part dedicated to these last cells (i.e. fibroblasts).

Author Response

Thank you for your comments. We revised the manuscript as you pointed out.

This is a very interesting review. about the interaction of MSCs and fibroblasts in wound healing. The paper is well written and every chapter is deeply thought and represented. However it's my opinion that is necessary to rewrite the chapter about the wound healing because in the decription of this one are completedy missed the contribution of some cells (for example mast cells). Besides about the interactions beetween the MSc and fibroblasts, is necessary to enlarge the part dedicated to these last cells (i.e. fibroblasts).

Thank you for your valuable comments. We focused on the interaction of MSCs and fibroblasts. So, we added the MSC functions on fibroblast under different conditions in Table. In addition, we added future prospects of MSCs in last section to enlarge the contribution of MSCs for cutaneous wound healing.

3.6. Future prospects of MSCs

Currently, research institutes and companies are developing methods for the mass culture of high-quality MSCs, but there are very few successful examples and cannot be applied in clinical settings. Therefore, researchers and physicians are carefully culturing in flasks one by one and administering them to individual patients. The establishment of a simple and accurate mass culture method for MSCs is one of the issues that must be solved immediately. Even when used for wound healing, MSCs will be able to be ad-ministered via intravenous infusion or topical injection without culture simultaneously [130]. A difficult administration method will cause confusion in the clinical setting, and itself makes difficult to further development. Ideally, MSCs should be administered in a manner that promotes rapid wound repair, similar to the administration of antimicro-bial agents. Furthermore, it is hoped that the exosomes and wound-healing substances secreted by MSCs could be used intravenous injection instead of using MSCs in itself, and these substances could provide to patients more conveniently due to development of self-administration drugs.

Round 2

Reviewer 1 Report

I thank the authors for their amendment of the original manuscript. A number of points have been improved; however, more corrections are needed. Notably, the first major point I have made was missed: it is important that the authors make clear that what they describe regarding the use of cultured MSCs for skin healing does not necessarily reflect what happens during natural wound healing: MSCs are an artefact of culture and the existence of identical cells in vivo has not been documented.

Comments for author File: Comments.docx

Author Response

Thank you for your time again. We revised the manuscript point by point. Fibroblasts originate in cutaneous tissue and are found in peripheral blood, wounds, and tissue remodeling. Also, a lot of study demonstrated that MSCs can proliferate cells and induce re-epithelialization and angiogenesis. So, it is important for us to understand and describe the interaction between fibroblast and MSCs. We rewrote the manuscript including the need or not for culturing.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors answered at my questions.

Author Response

Thank you for your time.