Peripheral Nerve Regeneration Using a Cytokine Cocktail Secreted by Skeletal Muscle-Derived Stem Cells in a Mouse Model

Round 1

Reviewer 1 Report

J of Clinical Med

jcm-1116838

 

Title:

Acceleration of Peripheral Nerve Regeneration Using a Cytokine Cocktail Secreted by Skeletal Muscle-Derived Stem Cells

Suggest: Acceleration of Peripheral Nerve Regeneration Using a Cytokine Cocktail Secreted by Skeletal Muscle-Derived Stem Cells in a Mouse Model

 

Keywords: severe peripheral nerve injury; regenerative medicine; therapeutic agent; therapeutic, adjunct; facilitation effect.

 

Abbreviations: 

skeletal muscle-derived stem cells (Sk-MSCs)

adult mouse serum (AMS)

Iscove’s modified Dulbecco’s medium (IMDM)

cytokine (CT) group

non-cytokine control (NT) group

 Good use of abbreviations. 

Purpose, Aims, or Hypothesis:

"In the present study, we investigated the peripheral nerve regeneration-accelerating capacity of Sk-MSC-derived cytokines using mouse Sk-MSCs. Therefore, this study is the first step in regenerative medicine without the use of stem cells, but with the use of stem cell functions (what are these), with the aim of achieving broader and easier therapeutic utility."

 

Introduction:  

The introduction should speak to the Purpose of the research.  Expansion of the introduction is suggested.

Further Suggestions:

1.The keywords could be words of a higher order medline category so your manuscript will be selected by more readers during a search.  Use terms such as peripheral nerve, skeletal muscle, cytokines, skeletal muscle-derived stem cells, mouse.

Title Suggestion: Acceleration of Peripheral Nerve Regeneration Using a Cytokine Cocktail Secreted by Skeletal Muscle-Derived Stem Cells in a Mouse Model

2. Title: It has become a custom that the animal model be included in the manuscript title. Mouse or murine would be adhering to this tradition.

3. Introduction: Line 55: “At present, Sk-MSC transplantation demonstrates maximum effects, with a >80% average of numerical and functional recovery following active cell engraftment and differentiation of Schwann cells, perineurial/endoneurial cells associated with vascular endothelial cells, and pericytes [8].”  Please explain what is meant by numerical and functional recovery.

 4.Line 63:

“In fact, in 63 human Sk-MSC transplantation experiments, it has been suggested that the total recovery 64 (75–100%) may include an approximately 60–80% contribution of the paracrine effects [11].”

Please explain what is meant by total recovery.

 5.These generalizations need to be explained with more specific examples. These requests are made because your purpose says: “In the present study, we investigated the peripheral nerve regeneration-accelerating capacity of Sk-MSC-derived cytokines using mouse Sk-MSCs. Therefore, this study is the first step in regenerative medicine without the use of stem cells, but with the use of stem cell functions, with the aim of achieving broader and easier therapeutic utility..

6. In the Introduction you have not yet specifically talked about: regeneration-accelerating capacity or stem cell functions. Accelaeration is different from extension/reconnection.

7.Line 94: stamps should probably read stumps.

8. Fig 1. NT, non-cytokine control group. What does that mean? Is Figure 1 showing only histo for Control Group.

 

9. Line 126: why was AMS/IMDM chosen for the CT rather than saline/IMDM or just 100% IMDM?

 

10. Table 1. Please define Cont-side and Op-side in the legend of the table. Also define the units of measure, N, mg. g., and *

 

11. Line 212. “This suggests that more recruitment of motor units oc-212 curred in plantar flexor muscles in the CT group according to the upstream nerve re-in-213 nervation/re-connection.” This may suggest as there are other possible reasons: larger motor units, faster reinnervation, better Schwann cell regeneration, better reorganization of reinnervation.

 

12. the legend for Fig 2 is not clear. Define Muscle Mass Recover (%) equation. Same for Tension Recovery (%).

 

13. Line 75. Please include the gender of the mice.

 

14. Fig 5 is not necessary. Shows same relationships as Fig 4.

 

15. some mice were lost in the final data. Tell us how the mice looked at 6 weeks post op. What happened during data collection that some mice are not reported?

 

16. Line 297. “Functional 297 and numerical recovery ratios of the present combination therapy using scaffold tube 298 bridging and cytokine cocktail administration was approximately 20–30%, which is lower 299 than a previous study where tube bridging and Sk-MSC transplantation was used (80–300 90% recovery) [8].

 

Line 196. Statistical analysis.  Remember from statistics class: When comparing interval data such as percentage data, a non-parametric test is appropriate (Wilcoxen for 2 groups).  The student’s t-test is not appropriate for the values of % or ratios. .

 

18. 5. Line 369. Conclusions

“This study demonstrated the first step of regenerative medicine without using stem cells, yet still employing its functions, aimed at a broader and easier adjuvant therapeutic utility as the cytokine cocktail, which exerts peripheral nerve regeneration accelerating ability for a variety of tissues in the body. Further studies to elucidate other specific factors  expressed by Sk-MSCs and development of a condensed method using this cytokine cocktail are necessary.”

 

The reader finds the conclusion is vague.  Please include the functions stem cells have which are carried by the cocktail into peripheral nerve regeneration.  The use of accelerating ability stretches the outcomes of the research design which does not test acceleration of nerve regeneration.  You would need more post op time points to measure acceleration. 

 

Author Response

Reviewer 1:

Response: We thank the reviewer for the overall positive comments. Our point-by-point-responses are as follows.

Acceleration of Peripheral Nerve Regeneration Using a Cytokine Cocktail Secreted by Skeletal Muscle-Derived Stem Cells

Suggest: Acceleration of Peripheral Nerve Regeneration Using a Cytokine Cocktail Secreted by Skeletal Muscle-Derived Stem Cells in a Mouse Model

 

Keywords: severe peripheral nerve injury; regenerative medicine; therapeutic agent; therapeutic, adjunct; facilitation effect.

Abbreviations: 

skeletal muscle-derived stem cells (Sk-MSCs)

adult mouse serum (AMS)

Iscove’s modified Dulbecco’s medium (IMDM)

cytokine (CT) group

non-cytokine control (NT) group

 Good use of abbreviations. 

Purpose, Aims, or Hypothesis:

• "In the present study, we investigated the peripheral nerve regeneration-acceleratingcapacity of Sk-MSC-derived cytokines using mouse Sk-MSCs. Therefore, this study is the first step in regenerative medicine without the use of stem cells, but with the use of stem cell functions (what are these), with the aim of achieving broader and easier therapeutic utility."

Response: We have re-written as follows.

In the present study, we investigated whether the peripheral nerve regeneration in vivo, such as axonal extension/reconnection, could be facilitated by repetitive administration of the mouse Sk-MSC-derived cytokines. Therefore, this study is the first step in regenerative medicine without the use of stem cells, but with the use of stem cell functions, with the aim of achieving broader and easier therapeutic utility. In other words, this is the stem cell therapy without cell transplantation, but use the administration of cytokine cocktails expressed by the stem cells.  

Introduction:  

• The introduction should speak to the Purpose of the research.  Expansion of the introduction is suggested.

Response: Please refer to above.

 

Further Suggestions:

1. The keywords could be words of a higher order medline category so your manuscript will be selected by more readers during a search.  Use terms such as peripheral nerve, skeletal muscle, cytokines, skeletal muscle-derived stem cells, mouse.

Response:  Thank you for the kind suggestion. In our understanding, Medline categorized the title words and the key words together. Therefore, we used to select key words different from the title words to get broader easier search results.  

2. Title Suggestion: Acceleration of Peripheral Nerve Regeneration Using a Cytokine Cocktail Secreted by Skeletal Muscle-Derived Stem Cells in a Mouse Model

Title: It has become a custom that the animal model be included in the manuscript title. Mouse or murine would be adhering to this tradition.

 

Response: We agree with the reviewer. We changed the title accordingly.

 

 

1. Introduction: Line 55: “At present, Sk-MSC transplantation demonstrates maximum effects, with a >80% average of numerical and functional recovery following active cell engraftment and differentiation of Schwann cells, perineurial/endoneurial cells associated with vascular endothelial cells, and pericytes [8].”  Please explain what is meant by numerical and functional recovery.

 

Response: We re-written as follows as suggested by the reviewer.

 

At present, Sk-MSC transplantation demonstrates maximum effects, with a >80% average of numerical (axon and myelinated fibers) and functional recovery (tetanic tension output via electrical stimulation) following active cell engraftment and differentiation of Schwann cells, perineurial/endoneurial cells associated with vascular endothelial cells, and pericytes [8].

 4.Line 63:

“In fact, in human Sk-MSC transplantation experiments, it has been suggested that the total recovery (75–100%) may include an approximately 60–80% contribution of the paracrine effects [11].”

Please explain what is meant by total recovery.

Response: We have rewritten this part in as follows..

In fact, in human Sk-MSC transplantation experiments, it has been suggested that the average total/final numerical/functional recovery (75–100%) during 12 weeks may include an approximately 60–80% contribution of the paracrine effects [11].

5. These generalizations need to be explained with more specific examples. These requests are made because your purpose says: “In the present study, we investigated the peripheral nerve regeneration-accelerating capacity of Sk-MSC-derived cytokines using mouse Sk-MSCs. Therefore, this study is the first step in regenerative medicine without the use of stem cells, but with the use of stem cell functions, with the aim of achieving broader and easier therapeutic utility.

Response: We added following sentence at the end of the Introduction to be generalized the purpose of this study.

In other words, this is the stem cell therapy without cell transplantation, but use the administration of cytokine cocktails expressed by the stem cells.

1. In the Introduction you have not yet specifically talked about: regeneration-accelerating capacity or stem cell functions. Accelaeration is different from extension/reconnection.

 

Response: We agree with the reviewer. Regeneration-accelerating capacity is somewhat indefinite description. Therefore, we have rewritten in as follows.

 

In the present study, we investigated whether the peripheral nerve regeneration in vivo, such as axonal extension/reconnection, could be facilitated by repetitive administration of the mouse Sk-MSC-derived cytokines.

7. Line 94: stamps should probably read stumps.

Response: Right, that is “stumps”, thus, we have changed.

1. Fig 1. NT, non-cytokine control group. What does that mean? Is Figure 1 showing only histo for Control Group.

 

Response: We have stated in the legend that “Typical cross-sectional view of the regenerated nerve of the NT group at position 1–4.”

 

In addition, description “non-cytokine control group” is re-written to “non-cytokine administered control group”.

1. Line 126: why was AMS/IMDM chosen for the CT rather than saline/IMDM or just 100% IMDM?

 

Response: Sk-MSCs basically showed active differentiation into myogenic cell lineage when the rapid decrease of serum concentration (20% to 5%) occurred. This process also diminished the cellular multipotency and this is undesirable action. When this method will be applied for the human therapy, we will use same manner (use human autologous serum).

 

 

1. Table 1. Please define Cont-side and Op-side in the legend of the table. Also define the units of measure, N, mg. g., and *

 

Response: We added “Cont-side, control-side; Op-side, Operation-side.” In the legend of the table. * is also added upper left of P.

We think that N, mg, and g have been defined in the Table 1. 

 

1. Line 212. “This suggests that more recruitment of motor units occurred in plantar flexor muscles in the CT group according to the upstream nerve re-innervation/re-connection.” This may suggest as there are other possible reasons: larger motor units, faster reinnervation, better Schwann cell regeneration, better reorganization of reinnervation.

 

Response: Basically, the force of skeletal muscle contraction is dependent on the number of recruitment motor units.  We would just like to describe the concept. In the present model, all motor units present in the right posterior limb were destroyed by the sciatectomy. So, there was zero tension. Then, the transected myelinated fibers of the alfa motoneuron have gradually re-connected, and the motor unit was re-established, and then the tension began to recover. At the transection cites of the sciatic nerve, it is possible that one axon reconnection means one motor unit recovery. In this process, above described factors by the reviewer are also involved. Thus, we think that the recruitment of motor units is comprehensive and correcting description.  

 

1. the legend for Fig 2 is not clear. Define Muscle Mass Recover (%) equation. Same for Tension Recovery (%).

 

Response: we added following equations in the legend.

 

“Muscle mass and tension recovery ratio was calculated as follows; (operated muscles mass/contralateral muscles mass) x 100, (tension output of operated muscles/tension output of contralateral muscles) x 100.”

 

1. Line 75. Please include the gender of the mice.

 

Response: We added “male and/or female” in the 2.1. Animals section.

 

 

1. Fig 5 is not necessary. Shows same relationships as Fig 4.

 

Response: The reviewer is right; these two graphs show the same trend. However, the amplitude of differences is almost double at the comparison based on the ratio. Thus, we would like to keep this figure 5.

 

1. some mice were lost in the final data. Tell us how the mice looked at 6 weeks post op. What happened during data collection that some mice are not reported?

 

Response: In the paragraph 2.5., we added following sentence as follows.

 

Four mice in the CT and 7 mice in the NT groups were directly used for the histology without the functional examination.

 

In addition, the number of animals used for the numerical analysis was added in the figure legend of Fig. 4 and 5.   

 

1. Line 297. “Functional and numerical recovery ratios of the present combination therapy using scaffold tube bridging and cytokine cocktail administration was approximately 20–30%, which is lower than a previous study where tube bridging and Sk-MSC transplantation was used (80–300 90% recovery) [8].

 

Line 196. Statistical analysis.  Remember from statistics class: When comparing interval data such as percentage data, a non-parametric test is appropriate (Wilcoxen for 2 groups).  The student’s t-test is not appropriate for the values of % or ratios.

 

Response: The reviewer is right. We re-examined by the Wilcoxon’s 2-pair non-parametric test for the percentage data, but the result was not different. We have re-written the Statistical analysis (para 2.7.) as follows.

 

Differences between the functional and numerical data of the two groups (CT- and NT-group) were tested using Student’s t-test (for the absolute values) and Wilcoxon non-parametric test (for the percentage value), and the significance level was set at p < 0.05. Values are expressed as the mean ± SE.

 

1. 5. Line 369. Conclusions

“This study demonstrated the first step of regenerative medicine without using stem cells, yet still employing its functions, aimed at a broader and easier adjuvant therapeutic utility as the cytokine cocktail, which exerts peripheral nerve regeneration accelerating ability for a variety of tissues in the body. Further studies to elucidate other specific factors  expressed by Sk-MSCs and development of a condensed method using this cytokine cocktail are necessary.”

The reader finds the conclusion is vague.  Please include the functions stem cells have which are carried by the cocktail into peripheral nerve regeneration.  The use of accelerating ability stretches the outcomes of the research design which does not test acceleration of nerve regeneration. 

Response: We re-organized the conclusion as follows, according to the reviewer’s suggestion.

This study demonstrated the first step of regenerative medicine without using stem cells, yet still employing its functions, aimed at a broader and easier adjuvant therapeutic utility as the cytokine cocktail derived from Sk-MSCs. This cocktail showed facilitating ability for the peripheral nerve regeneration based on the recovery of the reconnected number of axons and myelinated fibers and the tetanic tension output via electrical stimulation. This cocktail typically contained several angiogenesis and peripheral nerve regeneration relating factors, thus, possibly expect to the facilitation of the nerve-vascular regeneration in the variety of tissues. Further studies to elucidate other specific factors expressed by Sk-MSCs and development of a condensed method using this cytokine cocktail are necessary.  

19. You would need more post op time points to measure acceleration. 

Response: Based on our previous study [11], it was strongly suggested that the paracline effects of Sk-MSCs was most prominent during first 2-3 weeks of post-treatment. Then, this effect declined towards 4 weeks. Then, the effect remains almost the same level at 8 weeks thereafter. Therefore, we selected 6 weeks, and this is sufficient and appropriate period to evaluate the cytokine ability.  Based on this notion, we added following paragraph in the discussion (paragraph 3).   

Additionally, we selected the 6 weeks of recovery time in the present study. Based on our previous study [11], it was strongly suggested that the paracline effects of Sk-MSCs was most prominent during first 2-3 weeks of post-treatment. Then, this effect declined towards 4 weeks, and the effect remains almost the same level at 8 weeks thereafter. Therefore, we selected 6 weeks as the sufficient and appropriate period to evaluate the cytokine ability. In this regard, it is supposed that the first 2-3 weeks of post-nerve injury may be the most effective and important phase of this cytokine cocktail treatment. Therefore, this treatment should be started at acute phase and may be too late in the subacute phase.

Author Response File: Author Response.pdf

Reviewer 2 Report

English in this manuscript needs work. I would suggest an English (non-scientist) editor. Grammar seems to be fine but the order of many sentences needs work. Examples are below.

Abstract did not mention that GFP nerve graft was placed inside collagen tube. Please change.

Line 23: Last sentence. Remove "The" and change to: Mouse sciatic

line 35: Change "Should be" to "are likely" ....

Line 69: Change to read:  … we investigated the capacity of Sk-MSCs cytokines to enhance peripheral nerve regeneration in mice. 

Line 71: This study is not the first step in regenerative medicine etc. Reword please. Literature has numerous reports on the use of conditioned media, stem cell paracrine factors, exosome, etc., in regenerative medicine and tissue engineering. Are the authors confident and searched extensively to say no cytokine cocktail from stem cells has been used in nerve repair, in particular.

Line 85: Change Both side of the sciatic nerve to: Sciatic nerve (approximately 100 mm each) was removed from both sides of GFP ….

Line 88: Change to read: Preparation of the nerve regeneration model in wild-type mice was performed under inhalation anesthesia (isoflurane; Abbott, Tokyo, Japan). Body temperature was monitored by a rectal probe and maintained at …..  

Line 94: Change stamps to stumps.

Line 95: Nerve after transection retract. Authors said 6 mm gap in the abstract and 7 mm on line 93 methods. I am not clear what you mean by adjust the cut to 6 mm (Line 95). Please clarify. Nerve after transection retract more as you know. 

Line 105: nerve gap was created by transection and collagen tube. Make sure that is clear in the figure legend.

Figure 1 legend: clarify if the scale bar is 1 mm or 100 µm. You have listed both.

Line 116: Spell out Sk-34 and Sk-DN to add more clarity despite the citations.

Line 120: What do you mean by culture supernatant? Do you mean conditioned media? Change supernatant to culture media in Line 120 and keep it in line 122 to avoid confusion.

Line 136+137: Delete. You are already listed anesthesia and rectal temperature.

Table 1: List if the +/- are SD or SE. List what N means for the Tetanus. Is that Newton

Line 223: Figure 2 legend: List the meaning of NT and CT. List if the confidence interval is SD or SEM. See legend for figures 4+5.

Line 295-297: Please revise in view of earlier remark that others have used conditioned media

Was the SK-MSC cytokines cocktail tested on any stem cell to establish its neurogenic ability in vitro?

Please discuss (in the discussion) the effect of having a GFP nerve piece in the center of the collagen tube. Your cytokine comparison compared intact nerve, crushed nerve, and conditioned media. What is the effect of the GFP piece of nerve in the tube on the composition of the cytokine cocktail?

Please discuss the fact that you have treated an acute nerve injury rather than an old nerve injury and how is that going to affect clinical translation.

Author Response

Reviewer 2:

Response: We thank the reviewer for the overall positive comments. Our point-by-point-responses are as follows.

• English in this manuscript needs work. I would suggest an English (non-scientist) editor. Grammar seems to be fine but the order of many sentences needs work. Examples are below.

Response:  We sincerely appreciate the reviewer's review in English, and we have rewritten all of the sections as suggested. In this regard, we would like to say that we are not native speaker of English, thus, we always care for our English usage as much as possible, and we customarily send our manuscript to the professional English editor before submission.     

• Abstract did not mention that GFP nerve graft was placed inside collagen tube. Please change.

Response:  In this study, the GFP nerve graft is simply used as an indicator to correctly obtain histology sections 1 to 4 of Figure 1A. In other words, this is just the scale, and have no relation discussed in the present result.  Therefore, to avoid misapprehension, we would like to avoid describing GFP in the abstract.

• Line 23: Last sentence. Remove "The" and change to: Mouse sciatic

Response: We have done so.

• line 35: Change "Should be" to "are likely" ....

Response: We have done so.

• Line 69: Change to read:  … we investigated the capacity of Sk-MSCs cytokines to enhance peripheral nerve regeneration in mice.

Response: We changed the last paragraph of the Introduction to as follows.

In the present study, we investigated whether the peripheral nerve regeneration in vivo, such as axonal extension/reconnection, could be facilitated by repetitive administration of the mouse Sk-MSC-derived cytokines. Therefore, this study is the first step in regenerative medicine without the use of stem cells, but with the use of stem cell functions, with the aim of achieving broader and easier therapeutic utility. In other words, this is the stem cell therapy without cell transplantation, but use the administration of cytokine cocktails expressed by the stem cells. 

• Line 71: This study is not the first step in regenerative medicine etc. Reword please. Literature has numerous reports on the use of conditioned media, stem cell paracrine factors, exosome, etc., in regenerative medicine and tissue engineering. Are the authors confident and searched extensively to say no cytokine cocktail from stem cells has been used in nerve repair, in particular.

Response: We certainly want to say, “the first step” not “the first study”. Please refer to the re-organized last paragraph in the Introduction.  

• Line 85: Change Both side of the sciatic nerve to: Sciatic nerve (approximately 100 mm each) was removed from both sides of GFP ….

Response: We have done so.

• Line 88: Change to read: Preparation of the nerve regeneration model in wild-type mice was performed under inhalation anesthesia (isoflurane; Abbott, Tokyo, Japan). Body temperature was monitored by a rectal probe and maintained at …..  

Response:  We have done so.

• Line 94: Change stamps to stumps.

Response: We have done so.

• Line 95: Nerve after transection retract. Authors said 6 mm gap in the abstract and 7 mm on line 93 methods. I am not clear what you mean by adjust the cut to 6 mm (Line 95). Please clarify. Nerve after transection retract more as you know. 

Response: 0.5mm on both sides is suture marginal. Thus, we added this description in that portion.   

• Line 105: nerve gap was created by transection and collagen tube. Make sure that is clear in the figure legend.

Response: We believe that the details of the present sciatic nerve transection model are sufficiently described in the paragraph 2.2 and Figure 1A.   

• Figure 1 legend: clarify if the scale bar is 1 mm or 100 µm. You have listed both.

Response: We had listed both in the figure legend.

• Line 116: Spell out Sk-34 and Sk-DN to add more clarity despite the citations.

Response: We added the description in that portion as follows.

Sk-34 (skeletal muscle-derived CD34+/45-) and Sk-DN (skeletal muscle-derived CD34-/45-)

• Line 120: What do you mean by culture supernatant? Do you mean conditioned media? Change supernatant to culture media in Line 120 and keep it in line 122 to avoid confusion.

Response: With all due respect, we would like to say that “culture supernatant” is a established word in the biological dictionary.  

• Line 136+137: Delete. You are already listed anesthesia and rectal temperature.

Response: The first description is intended for a preparation of the nerve regeneration model, while the second (suggested part) is a functional measure. There are fundamentally different experiment. Thus, we explained again.  

• Table 1: List if the +/- are SD or SE. List what N means for the Tetanus. Is that Newton

Response: Table 1 legend is totally re-organized as follows.

NT, non-cytokine administered control group; CT, cytokine administered group; Cont-side, control-side; Op-side, Operation-side. N=Newton. Values are expressed as mean ± SE.

• Line 223: Figure 2 legend: List the meaning of NT and CT. List if the confidence interval is SD or SEM. See legend for figures 4+5.

Response:  We have done so.

• Line 295-297: Please revise in view of earlier remark that others have used conditioned media

Response: The previous study is a stem cell transplantation study, and the present study is a cytokine administered study without cell transplantation.  Culture media used during cell expansion is the same in both experiments (IMDM/20% FBS). However, in the cell transplantation study, the expanded cells were essentially washed and suspended by serum-free culture media and then transplanted to prevent GVHD. Moreover, we believe that the reader's conduct to the series of our work is also the meaning of this study.  

• Was the SK-MSC cytokines cocktail tested on any stem cell to establish its neurogenic ability in vitro?

Response: Actually, no we didn’t. We basically think that the in vitro study situated very basic of the transrational study in vivo. Thus, we believe that there is not inevitable experiment. A large number of studies, which logically connected in vitro results and the availability of transplantation. However, it is also the fact that a large number of in vitro studies published without in vivo confirmation data. We think that the latter case is not mean anything as the therapeutic regenerative study, and in vitro evidence is not indispensable.   

• Please discuss (in the discussion) the effect of having a GFP nerve piece in the center of the collagen tube. Your cytokine comparison compared intact nerve, crushed nerve, and conditioned media. What is the effect of the GFP piece of nerve in the tube on the composition of the cytokine cocktail?

Response: Please refer to the above response #2. The GFP nerve graft is simply used as an indicator to correctly obtain histology sections 1 to 4 of Figure 1A. Of course, we have published the contribution of the GFP graft as the supplier of Schwann cells, but the effect of axon and myelinated fibre growth is small and not significantly different than that of the media group [8]. In addition, there is the same in the CT and NT group, and the difference is whether or not adding cytokine cocktail. However, we added following paragraph in the discussion (second para).

In the present study, we used the GFP nerve graft in the center of the collagen tube consistently in the NT and CT group, as an indicator to correctly obtain histology sections 1 to 4 of Figure 1A. In regard to the GFP nerve graft, we reported that the GFP graft limitedly contributed as the Schwann cell supplier, but the effect of axon and myelinated fiber growth is small and not significantly different than that of the media group [8]. Therefore, we think that this is not the main factor of the present study.     

• Please discuss the fact that you have treated an acute nerve injury rather than an old nerve injury and how is that going to affect clinical translation.

Response: Based on our previous study [11], it was strongly suggested that the paracline effects of Sk-MSCs was most prominent during first 2-3 weeks of post-treatment. Then, this effect declined towards 4 weeks. Then, the effect remains almost the same level at 8 weeks thereafter. Therefore, we selected 6 weeks, and this is sufficient and appropriate period to evaluate the cytokine ability.  Based on this notion, we added following paragraph in the discussion (paragraph 3).   

Additionally, we selected the 6 weeks of recovery time in the present study. Based on our previous study [11], it was strongly suggested that the paracline effects of Sk-MSCs was most prominent during first 2-3 weeks of post-treatment. Then, this effect declined towards 4 weeks, and the effect remains almost the same level at 8 weeks thereafter. Therefore, we selected 6 weeks as the sufficient and appropriate period to evaluate the cytokine ability. In this regard, it is supposed that the first 2-3 weeks of post-nerve injury may be the most effective and important phase of this cytokine cocktail treatment. Therefore, this treatment should be started at acute phase and may be too late in the subacute phase.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors describe a novel therapeutic approach to peripheral nerve repair. Strong evidence was previously reported, including by several co-authors of the present study, that skeletal muscle cells (Sk-MSCs), and particularly via their paracrine effects, may have high potential to facilitate peripheral nerve repair (Tamaki et al. 2014, PLoS One; Tamaki et al. 2016, PLoS One). Here they evaluate cytokine treatment (CT) of Sk-MSCs-derived factors and compare it to non-treatment (NT) in a mouse nerve resection model. They found that CT increased the tetanic tension output of the plantar flexor muscles, and increased the absolute number of axons and their myelination in the damaged nerve, compared to NT. They also identified 17 growth factors and cytokines in the CT cocktail, which presumably contributed to its beneficial effects, and they were grouped into those that were uniquely found in the CT cocktail, those that were also endogenously found in damaged nerve compared to undamaged nerve, and those that were also endogenously found in both damaged and undamaged nerve equally. Overall, the study is well designed and led to interesting and clinically-relevant findings. Several minor requested changes to the presentation of the data are as follows:

1. In the Methods, it is stated that 10 mice received CT and 15 mice received NT. However, Table 1 indicates that data only represent 6 mice for CT and 8 mice for NT. Other figures do not indicate the number of mice represented in each group. The exact group sizes for each type of analysis and an explanation of how they were chosen should be included.
2. It is unclear if/how the levels of various factors presented in Figure 6 were normalized, as the values are arbitrary (pixels), and the samples were derived both in vitro (CT cocktail) and in vivo (normal or damaged nerve tissue). Were the total protein levels of each sample type the same? Some additional details should be provided in the Methods or figure legend to allow the reader to interpret the significance of the much higher levels found in the CT cocktail compared to both normal and damaged nerve tissue for most of the factors shown. Since growth factors and cytokines have notoriously short half-lives in vivo, it would be interesting in a future study to collect the nerve graft at different time points following CT or NT treatment to better evaluate the relative levels of cytokines provided by CT compared to the normal physiological response to damage (in NT).
3. In the Introduction and/or Discussion sections, the authors should elaborate on the potential clinical benefits of an acellular therapy (e.g., CT) compared to cell (e.g., Sk-MSCs) transplantation therapy for peripheral nerve repair. The current discussion is limited to “broader and easier therapeutic utility” and that it “makes clinical application much easier than cell transplantation therapy in every country”, though much more specific potential benefits exist.

Author Response

Reviewer 3:

The authors describe a novel therapeutic approach to peripheral nerve repair. Strong evidence was previously reported, including by several co-authors of the present study, that skeletal muscle cells (Sk-MSCs), and particularly via their paracrine effects, may have high potential to facilitate peripheral nerve repair (Tamaki et al. 2014, PLoS One; Tamaki et al. 2016, PLoS One). Here they evaluate cytokine treatment (CT) of Sk-MSCs-derived factors and compare it to non-treatment (NT) in a mouse nerve resection model. They found that CT increased the tetanic tension output of the plantar flexor muscles, and increased the absolute number of axons and their myelination in the damaged nerve, compared to NT. They also identified 17 growth factors and cytokines in the CT cocktail, which presumably contributed to its beneficial effects, and they were grouped into those that were uniquely found in the CT cocktail, those that were also endogenously found in damaged nerve compared to undamaged nerve, and those that were also endogenously found in both damaged and undamaged nerve equally. Overall, the study is well designed and led to interesting and clinically-relevant findings. Several minor requested changes to the presentation of the data are as follows:

Response: We thank the reviewer for the overall positive comments. Our point-by-point-responses are as follows.    

1. In the Methods, it is stated that 10 mice received CT and 15 mice received NT. However, Table 1 indicates that data only represent 6 mice for CT and 8 mice for NT. Other figures do not indicate the number of mice represented in each group. The exact group sizes for each type of analysis and an explanation of how they were chosen should be included.

 

Response: We thank the reviewer. This is an important issue we missed. We added the usage of the number of animals in each relating part such as Method (para 2.5) and Figure legend (Fig.4).

  

1. It is unclear if/how the levels of various factors presented in Figure 6 were normalized, as the values are arbitrary (pixels), and the samples were derived both in vitro (CT cocktail) and in vivo (normal or damaged nerve tissue). Were the total protein levels of each sample type the same? Some additional details should be provided in the Methods or figure legend to allow the reader to interpret the significance of the much higher levels found in the CT cocktail compared to both normal and damaged nerve tissue for most of the factors shown.

Response: We added following sentences in the method (Para 2.6).

 

All samples were normalized by protein concentration, and applied appropriate amount following the instruction of the Array Kit. Relative expression of each samples was expressed by pixel intensity based on the reference spot.

 

1. Since growth factors and cytokines have notoriously short half-lives in vivo, it would be interesting in a future study to collect the nerve graft at different time points following CT or NT treatment to better evaluate the relative levels of cytokines provided by CT compared to the normal physiological response to damage (in NT).

 

Response: We appreciate the excellent suggestion, and we agree with the reviewer. We would like to try the suggested experiment in the near future.

 

1. In the Introduction and/or Discussion sections, the authors should elaborate on the potential clinical benefits of an acellular therapy (e.g., CT) compared to cell (e.g., Sk-MSCs) transplantation therapy for peripheral nerve repair. The current discussion is limited to “broader and easier therapeutic utility” and that it “makes clinical application much easier than cell transplantation therapy in every country”, though much more specific potential benefits exist.

 

Response:  We thank the reviewer for the constitutive understanding of this work. However, as this issue is somewhat ethical, thus, we would like to moderate the description about this issue. We are planning the same experiment using human Sk-MSCs to establish the clinical application of this method. thus, we would like to gradually progress this issue through the future study.  

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Maki and colleagues report on the effects of a cytokine cocktail produced by muscle-derived stem cells (MSC) on the regenerative outcome after a severe nerve injury, i.e. the transection of the sciatic nerve with removal of a nerve segment and reconnection of the 2 stumps via a collagen tube, where a nerve graft is implanted.

They compare non treated, operated mice with a group of cytokine-treated, operated animals in terms of recovery of tension, axonal number and myelinated fibers number, finding out a global improvement. They propose the administration of the cytokine cocktail as a useful strategy to accelerate nerve recovery of function.

 

Reviewer’s concerns. The overall improvement in nerve recovery following cytokine cocktail treatment appears rather limited, and the underlying molecular mechanisms are not addressed in a satisfactory way.

Table 1: the difference in terms of absolute muscle mass between operated CT and NT is not statistically significant, but it becomes so when expressed relative to the initial mass, although the improvement is rather limited. Why did authors speak about a recovery in muscle mass? They are not monitoring muscle mass during the time course of the regeneration process, as just a single time point has been analysed (6 weeks). Couldn’t it be that the treatment affects nerve degeneration, thus reducing muscle atrophy?

What about the nerve graft? What are the effects of the cytokine treatment on it? No information is reported in the manuscript. The GFP-positive nerve graft has been shown at the beginning, but then somehow missed. No imaging during the time-course of the treatment, nor at the conclusion after 6 weeks is shown.

 

Fig. 4 Scale reported in y axis: what numbers refer to? The mean number of axons/slice??

 

The main concerns relate to the analysis of the cytokine cocktail.

Authors have compared by antibody arrays the cytokine composition/levels in the superatant of MSC cells in culture (without any stimuli, no injury) with that in sciatic nerve lysates in the absence or following injury. I think that the 2 models are totally different and cannot therefore be compared in terms of expression levels. How the levels of a cytokine secreted in a cell supernatant can be compared with those in a total nerve lysate? One can only compare cytokine levels between damaged nerve vs normal nerve. In addition, as cytokines are secreted molecules, they can be ‘lost’ in experiments employing cell lysates.

What ‘Pixel intensity’ in the y axis of Fig. 6 stands for?

Line 250: ‘Group 3: factors unchanged………but increased in Sk-MSC.’ Why increased? You mean more abundant? More expressed? MSC were not exposed to any stimuli/injury.

The discussion related on the last part of the study is very poor, just a list of molecules is provided. Having found them by the proteome array does not imply that they are all involved/important in the recovery of function of the nerve. What does this cocktail do in vivo? Does it target the nerve graft?

 

Minor points
-Lines 176, 178: ‘crush’ instead of ‘crash’

 

 

 

Author Response

For the reviewer 1:

 

We appreciate valuable input and helpful comments from the reviewer.

 

For this revision, first of all, we appreciate your understanding that the goal of this study is the establishment of the peripheral nerve regeneration accelerating agent. Then, this study was carried out as an objective of a wider and easier therapeutic utility such as the cytokine cocktail without using the stem cell transplant itself. Therefore, achieving sufficient recovery from this severe differential model of sciatic nerve transection is not a goal, and this is used as a fair model for assessing nerve recovery ability. Of course, we also make an effort to achieve our main goal, which is regenerative therapy to obtain sufficient recovery of severe nerve transection gap damage using stem cell transplantation in the previous study (please refer to ref 8-11).

This context was set out in the first paragraph of the discussion.

In this regard, the main focus of this study is whether the cytokine cocktail treatment is effective for the nerve regeneration or not. The following point-by-point responses are therefore based on this concept.

 

Reviewer’s concerns.

1. The overall improvement in nerve recovery following cytokine cocktail treatment appears rather limited, and the underlying molecular mechanisms are not addressed in a satisfactory way.

Response:

As suggested by the reviewer, the nerve regenerative effects of cytokine treatment is rather limited in comparison with our previous report using the direct injection of the Sk-MSCs transplantation (see ref. 8-11, 17). However, from another viewpoint, this has a 2-fold greater recovery than solo scaffold bridging treatment (NT group) alone, and this facilitation rate may be sufficient in clinical practice. This context was set out in the first paragraph of the discussion.

For the underlying molecular mechanisms, basically, this study is in vivo therapeutic study, thus, there is no way to analyze the administered cytokines induced molecular events in the recipient cells/tissues. For this analysis, in vitro cellular experiment with knockout/knock-in, addition of inhibiter/accelerator/mediator or agonist/antagonist are necessary for each 17 cytokines, which was found in the present study, but this is not the purpose of this study. Establishing a suitable cell culture system, like motor/sensory neurons and/or Schwann cells, is also almost impossible.  From another perspective, the present study clearly showed that the acceleration of motor nerve axonal growth and myelination in vivo were induced by the cytokine cocktail, and

these included 17 mainly enhanced cytokines.      

 

2. Table 1: the difference in terms of absolute muscle mass between operated CT and NT is not statistically significant, but it becomes so when expressed relative to the initial mass, although the improvement is rather limited. Why did authors speak about a recovery in muscle mass? They are not monitoring muscle mass during the time course of the regeneration process, as just a single time point has been analysed (6 weeks). Couldn’t it be that the treatment affects nerve degeneration, thus reducing muscle atrophy?

Response:

Based on the data of our previous human Sk-MSCs transplantation study (ref. 11), we suggest that this difference is due to reduced muscle atrophy, because the recovery of muscle mass could be started after 6 weeks of the recovery term.

The reason for the lack of muscle mass comment, it was just not significant, although, the tendency to prevention of muscle atrophy was observed.     

 

3. What about the nerve graft? What are the effects of the cytokine treatment on it? No information is reported in the manuscript. The GFP-positive nerve graft has been shown at the beginning, but then somehow missed. No imaging during the time-course of the treatment, nor at the conclusion after 6 weeks is shown.

Response:

This is simply used as the marker of the central portion of the nerve conduit. Fundamentally, the positive effect of nerve regeneration could be expected on this therapy, but this is common for both the NT and CT groups. Thus, it was convenient to get nervous peace correspond to section numbers in Fig. 1. At anyhow, we added following sentence in the line 95-96.

“This was used as an indicator to obtain the histological sections at position 1-4 in Figure 1A and C-F.”     

4. 4 Scale reported in y axis: what numbers refer to? The mean number of axons/slice?? 

Response:

Yes, there are the mean number of axons and/or myelinated fibers/sections. We thank the kind suggestion of the reviewer and have added “/section” in the Figure 4 and its Figure legend. 

5. The main concerns relate to the analysis of the cytokine cocktail. Authors have compared by antibody arrays the cytokine composition/levels in the superatant of MSC cells in culture (without any stimuli, no injury) with that in sciatic nerve lysates in the absence or following injury. I think that the 2 models are totally different and cannot therefore be compared in terms of expression levels. How the levels of a cytokine secreted in a cell supernatant can be compared with those in a total nerve lysate? One can only compare cytokine levels between damaged nerve vs normal nerve. In addition, as cytokines are secreted molecules, they can be ‘lost’ in experiments employing cell lysates.

Response:

Yes, the reviewer is right, there are essentially different model. The sciatic nerve crush injury model is a conformation model to determine which cytokine is closely linked to nerve regeneration in this analysis.

  On the other hand, the Sk-MSCs secreted cytokines were determined by the comparison between the supernatant of one day culture of Sk-MSCs/(20% adult mouse serum/IMDM) and no cell (20% adult mouse serum/IMDM). Then, there were analyzed together by the same Proteome Array kit following standard instruction with normalization and application.

These contexts were set out in the paragraph “2.6. Protein analysis for the cytokine cocktail” in the Experimental Section, and we also added following sentence in the last of this paragraph.

 

“The sciatic nerve crush injury model was used as a conformation model to determine which cytokine was closely linked to nerve regeneration in this analysis.”

 

Additionally, we did not analyze cell lysates of Sk-MSCs to avoid a defocus of this study. This is because our aim is absolutely a therapeutic application of the cytokine cocktail purely secreted by the Sk-MSCs.

6. What ‘Pixel intensity’ in the y axis of Fig. 6 stands for?

Response:

This is the commonly used unit of the currently used array kit. We realize that this corresponds to a density of antibody reactions.   

7. Line 250: ‘Group 3: factors unchanged………but increased in Sk-MSC.’ Why increased? You mean more abundant? More expressed? MSC were not exposed to any stimuli/injury.

Response:

Extraction of Sk-MSC from skeletal muscle then cultivation itself is a great stimulation for the stem cells. In this regard, we determined Sk-MSCs secreted specific cytokines by the comparison between Sk-MSCs/ IMDM containing 20% AMS culture supernatant and only IMDM containing 20% AMS using Proteome Array kit.

8. The discussion related on the last part of the study is very poor, just a list of molecules is provided. Having found them by the proteome array does not imply that they are all involved/important in the recovery of function of the nerve. What does this cocktail do in vivo? Does it target the nerve graft?

Response:

The target is absolutely the nerve axons (transected stumps), Schwann cells and perineurial/endoneurial cells. Angiogenesis/vasculogenesis relating cells, such as endothelial cells, pericyte, and vascular smooth muscle cells are also a target, because there are all closely relating to the nerve regeneration. That is why we have included the following descriptions in the second last paragraph.  

“There could be effects activating axonal growth of transected nerve stumps, and with nerve regeneration surrounding related cells, such as Schwann cells and perineural/endoneurial cells. Cells linked to angiogenesis/vasculogenesis, such as endothelial cells, pericyte and vascular smooth muscle cells, were also affected for their proliferation/differentiation.”

9. Minor points
   -Lines 176, 178: ‘crush’ instead of ‘crash’

Response:

We appreciate your suggestion. We replaced crash to crush in the text.

Reviewer 2 Report

This is an interesting study showing how the peripheral nerve regeneration can be accelerated using cytokine cocktail secreted by skeletal muscle-derived stem cells. However, the data presentation/analysis is not convincing. The major issues are:

 

Is there any specific reason behind using 20% fetal calf serum or adult mouse serum in the IMDM? isn't this too high?

 

How were the operated sciatic nerves isolated? Were the mice perfused? Please provide the details. Similarly, details for the washing with a graded sucrose series will be appreciated.

 

"Number of axons and myelinated fibers" - how were these quantified? The images presented are not of the grade to do such quantifications. Please present representative images wherever required. Photographs presented to show axon and myelinated fiber formation in regenerated sciatic nerves are not clear and difficult to interpret. Authors may need to quantify the regenerated axons, myelinated and non-myelinated fibers, which can be done with toluidine blue stained images. The ideal with be with EM photographs.

 

How was the pixel density standardized?

 

Authors may need to perform motor and sensory behavioral assessments to show the functional recovery of the regenerating nerves. It’s crucial.

Author Response

For the reviewer 2:

 

We appreciate valuable input and helpful comments from the reviewer.

 

For this revision, first of all, we appreciate your understanding that the goal of this study is the establishment of the peripheral nerve regeneration accelerating agent. Then, this study was carried out as an objective of a wider and easier therapeutic utility such as the cytokine cocktail without using the stem cell transplant itself. Therefore, achieving sufficient recovery from this severe differential model of sciatic nerve transection is not a goal, and this is used as a fair model for assessing nerve recovery ability. Of course, we also make an effort to achieve our main goal, which is regenerative therapy to obtain sufficient recovery of severe nerve transection gap damage using stem cell transplantation in the previous study (please refer to ref 8-11).

This context was set out in the first paragraph of the discussion.

In this regard, the main focus of this study is whether the cytokine cocktail treatment is effective for the nerve regeneration or not. The following point-by-point responses are therefore based on this concept.

Reviewer’s concerns.

This is an interesting study showing how the peripheral nerve regeneration can be accelerated using cytokine cocktail secreted by skeletal muscle-derived stem cells. However, the data presentation/analysis is not convincing. The major issues are:

1. Is there any specific reason behind using 20% fetal calf serum or adult mouse serum in the IMDM? isn't this too high?

Response:

This is inevitable condition of the Sk-MSCs culture.

2. How were the operated sciatic nerves isolated? Were the mice perfused? Please provide the details. Similarly, details for the washing with a graded sucrose series will be appreciated.

Response:

The sciatica nerve was freshly isolated to be kept high emitting GFP. (Fixation extremely reduced the GFP emission) and extended and pasted on the paper filter. Then, photographed as in Figure 1B. Then, immersed in the 4% PFA overnight.

Graded sucrose series is a process of substitution of tissue fluid. This is effective for the prevention of ice crystal formation and tissue cracking. These issues are represented in the subsection 2.5 in the Method.     

3. "Number of axons and myelinated fibers" - how were these quantified? The images presented are not of the grade to do such quantifications. Please present representative images wherever required. Photographs presented to show axon and myelinated fiber formation in regenerated sciatic nerves are not clear and difficult to interpret. Authors may need to quantify the regenerated axons, myelinated and non-myelinated fibers, which can be done with toluidine blue stained images. The ideal with be with EM photographs.

Response:

Firstly, we detected axon and the myelination by protein level using specific antibody (N200 and MBP) and immunohistochemistry. This has a benefit when partial and unavoidable histological alteration or damage has been induced by the slice sample. Of course, the reviewer is right, detection with toluidine blue with resin section and EM is better method. On the other hand, however, these are useful to limited area. In this study, all axons and myelinated fibers were detected in the whole sciatic nerve to assess absolute recovery. In addition, the tiling image method using higher magnification photograph was used in the present study (Fig. 1C-F, Fig. 3). In this way, axon/myelin could be counted at the higher magnification. However, the editor is correct, these digital issues are a bit confusing to readers. Thus, we added new photographs (Analytical image of N200 and MBP) in Fig. 3 and explanation as follows in the end of paragraph 2.5. Figure legend of Fig. 3 was also done.

  

“All axons and myelinated fibers were detected in the whole sciatic nerve to assess absolute recovery using the tiling image method of mbf with higher magnification photograph.”

4. How was the pixel density standardized?

Response:

This is the commonly used unit of the currently used array kit. We realize that this corresponds to a density of antibody reactions.

 

5. Authors may need to perform motor and sensory behavioral assessments to show the functional recovery of the regenerating nerves. It’s crucial.

Response:

Again, the reviewer is correct, the evaluation of behavioral functional recovery is important. In this respect, please see our first response above, followed by the following.

• The objective of this study is to determine whether or not the cytokine cocktail secreted by Sk-MSC was useful for peripheral nerve regeneration.
• In the previous Sk-MSCs transplantation studies, we have achieved the favorable nerve regeneration results, such as close to 90% numerical, morphological and functional recovery in the sciatic nerve long gap transection model, and this is the background of this study.
• Through the above experiments, it has also been established that the paracrine effects of Sk-MSC share some of the reason for the favorable outcome.
• As a result, fully favorable recovery and/or effect is not expectable in the present study, and we have to clarify the effect of a treatment more properly within the small amplitude. In some instances, no recovery has been achieved.
• Generally, quantifying behavioral assessment for mice is not so easy, and it is difficult to detect small differences.
• In this regard, the analysis of downstream muscle tension by electrical stimulation of the upper part of the damaged nerve is entirely quantitative and correct, and it depends entirely on recovery of the upstream nerve.
• There are the reasons why we decided on the functional assessment.

Round 2

Reviewer 1 Report

Most of the concerns emerged during the first revision have not been adequately addressed by the authors in the reply to the Reviewer. This Reviewer still believes that the study lacks a mechanistic insight on the effects of the cytokine cocktail described in the study. The justification provided that 'For the underlying molecular mechanisms, basically, this study is in vivo therapeutic study, thus, there is no way to analyze the administered cytokines induced molecular events in the recipient cells/tissues. For this analysis, in vitro cellular experiment with knockout/knock-in, addition of inhibiter/accelerator/mediator or agonist/antagonist are necessary for each 17 cytokines, which was found in the present study, but this is not the purpose of this study. Establishing a suitable cell culture system, like motor/sensory neurons and/or Schwann cells, is also almost impossible......' appears rather naive.

Moreover, the analysis of the effects of cytokine administration in vivo is incomplete, as a single time point (6 weeks) has been considered during nerve repair. 

Reviewer 2 Report

Not satisfactory