Reviewer ’s report  
 
Title: The HO -1-expressing bone mesenchymal stem cells protects intestine from ischemia and  
reperfusion injury  
 
Version: 2 Date: 04 Dec 2018  
 
Reviewer: Kaatje Lenaerts  
 
Reviewer's report:  
 
General:  
- The manuscript describes a study on the protective potential of HO -1-expressing BMSC cells 
on the development of intestinal IR injury and survival in mice. Data are clearly presented; the 
experiments seem to be performed adequately. A clear protective of  the therapy is shown; 
however the novelty is limited since both BMSC cells and HO -1 has been shown to be protective 
in earlier studies. The effect of the combination is not clearly different from the effect of BMSC 
alone.  
- Incorrect use of scientific Eng lish throughout the manuscript text. Authors should ask a native 
English speaker from the biomedical field to revise the manuscript text.  
- Grammar mistakes are present throughout manuscript text.  
 
 
Background.  
- Intestinal IR is an important disease entit y with a high mortality but stating that "Ischemia 
reperfusion injury (I/R) is one of the major causes of high morbidity, disability, and mortality in 
the world." is too strong.  
- Background section is not focused, and a lot of duplicate information is pre sent.  
 
 
 
 
 
Materials & Methods:  
- Not clear why male SD rats are used in the study. As source of BMSCs? Seems not as they 
were purchased from a company.  
- Which primers were used? Refer to table where the sequences are shown.  
- Include power calculation to ju stify the number of animals per group used.  
- How where mice kept stable in during intestinal ischemia? E.g. was their temperature 
monitored? Was fluid administered? How was the abdomen closed? Etc.  
- Which intervention was performed in the sham group? Did  mice receive also PBS instead of 
BMSC?  
- Were 5 mice per group used for survival analysis and 5 mice for analyses after 24h? Hence, in 
n=10 in total per group? Or where 80 animals used instead of 40?  
- Which parameters were monitored during survival? Wher e animal welfare sheets used? Provide 
details on the health status of animals, and not only survival data.  
- Who performed the analysis of small intestinal injury? It is stated that it was performed by 
Lenaerts et al [16]. But reference 16 is an article by  Ceulemans et al. It could be that the authors 
mean that it was performed according to the article by Ceulemans et al. This should be correctly 
indicated.  
- Information on IR injury scoring is lacking. How many fields were scored at which 
magnification etc .? Was the scoring performed blindly?  
- Which statistical test was used to find significant differences? In the manuscript, only the post 
hoc test is mentioned.  
 
 
 
 
 
 
 
 
Results  
- Authors should explain the term MOI, p8, l1  
- Figure 1A. information on how many sam ples that were analyzed is missing.  
- Not clear why nuclear expression of GFP is higher in de BMSC/HO -1 group. What does this 
indicate?  
- It appears from Figure 1C a twofold increase was observed. This seems not a dramatic increase, 
as is stated on p8 l12/ 13 
- Correct labels should be used in all figures, since it is not clear what model and vector stands 
for. Use the terms sham - IR - BMSC - BMSC/HO -1 correctly. Also, in the manuscript text and 
legends this should be used consistently.  
- Again, increases in HO expression (Figure 3) are not dramatic.  
- Information on the plasma levels of the cytokines is missing. It is important to assess the 
potential of BMSCs on decreasing these systemic cytokines, as they play a large role in 
morbidity  of IR injury.  
- Information on the localization of the BMSCs in the injured tissue is lacking. As they are GFP -
positive, they can be tracked which would add clinically relevant information to the manuscript. 
Moreover, there phenotype could be assessed, to  evaluate their plasticity.  
- Are differences between Vector and HO -1 not significant in figure 4? What is the added value 
of HO -1? Also, in survival there were no differences between BMSC and BMSC -HO-1. 
 
Discussion  
- Survival rates were not significantly different between BMSC and BMSC -HO-1. This is 
incorrectly stated at p10, l38 -42. 
- MDA levels are discussed, however, there are no results presented on MDA levels.  
- Again, what is the added value of BMSC -HO-1 over BMSC alone, according to cytokine 
measur ements? It seems not correctly described on p11, l9 -11. 
- Data on HO -1 should be discussed in more detail, since it is already known that HO -1 has 
protective effects in intestinal IR. What is the added value of BMSC -HO-1 over HO -1? 
- It should also be disc ussed what these findings could mean for patients suffering from intestinal 
IR. Can such therapies be translated to the human situation?  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary  controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any stati stics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five year s received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in a ny way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding , or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you  can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competi ng interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Acces s Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: The HO -1-expressing bone mesenchymal stem cells protects intestine from ischemia and  
reperfusion injury  
 
Version: 2 Date: 04 Dec 2018  
 
Reviewer: Kaatje Lenaerts  
 
Reviewer's report:  
 
General:  
- The manuscript describes a study on the protective potential of HO -1-expressing BMSC cells 
on the development of intestinal IR injury and survival in mice. Data are clearly presented; the 
experiments seem to be performed adequately. A clear protective of  the therapy is shown; 
however the novelty is limited since both BMSC cells and HO -1 has been shown to be protective 
in earlier studies. The effect of the combination is not clearly different from the effect of BMSC 
alone.  
- Incorrect use of scientific Eng lish throughout the manuscript text. Authors should ask a native 
English speaker from the biomedical field to revise the manuscript text.  
- Grammar mistakes are present throughout manuscript text.  
 
 
Background.  
- Intestinal IR is an important disease entit y with a high mortality but stating that "Ischemia 
reperfusion injury (I/R) is one of the major causes of high morbidity, disability, and mortality in 
the world." is too strong.  
- Background section is not focused, and a lot of duplicate information is pre sent.  
 
 
 
 
 
Materials & Methods:  
- Not clear why male SD rats are used in the study. As source of BMSCs? Seems not as they 
were purchased from a company.  
- Which primers were used? Refer to table where the sequences are shown.  
- Include power calculation to ju stify the number of animals per group used.  
- How where mice kept stable in during intestinal ischemia? E.g. was their temperature 
monitored? Was fluid administered? How was the abdomen closed? Etc.  
- Which intervention was performed in the sham group? Did  mice receive also PBS instead of 
BMSC?  
- Were 5 mice per group used for survival analysis and 5 mice for analyses after 24h? Hence, in 
n=10 in total per group? Or where 80 animals used instead of 40?  
- Which parameters were monitored during survival? Wher e animal welfare sheets used? Provide 
details on the health status of animals, and not only survival data.  
- Who performed the analysis of small intestinal injury? It is stated that it was performed by 
Lenaerts et al [16]. But reference 16 is an article by  Ceulemans et al. It could be that the authors 
mean that it was performed according to the article by Ceulemans et al. This should be correctly 
indicated.  
- Information on IR injury scoring is lacking. How many fields were scored at which 
magnification etc .? Was the scoring performed blindly?  
- Which statistical test was used to find significant differences? In the manuscript, only the post 
hoc test is mentioned.  
 
 
 
 
 
 
 
 
Results  
- Authors should explain the term MOI, p8, l1  
- Figure 1A. information on how many sam ples that were analyzed is missing.  
- Not clear why nuclear expression of GFP is higher in de BMSC/HO -1 group. What does this 
indicate?  
- It appears from Figure 1C a twofold increase was observed. This seems not a dramatic increase, 
as is stated on p8 l12/ 13 
- Correct labels should be used in all figures, since it is not clear what model and vector stands 
for. Use the terms sham - IR - BMSC - BMSC/HO -1 correctly. Also, in the manuscript text and 
legends this should be used consistently.  
- Again, increases in HO expression (Figure 3) are not dramatic.  
- Information on the plasma levels of the cytokines is missing. It is important to assess the 
potential of BMSCs on decreasing these systemic cytokines, as they play a large role in 
morbidity  of IR injury.  
- Information on the localization of the BMSCs in the injured tissue is lacking. As they are GFP -
positive, they can be tracked which would add clinically relevant information to the manuscript. 
Moreover, there phenotype could be assessed, to  evaluate their plasticity.  
- Are differences between Vector and HO -1 not significant in figure 4? What is the added value 
of HO -1? Also, in survival there were no differences between BMSC and BMSC -HO-1. 
 
Discussion  
- Survival rates were not significantly different between BMSC and BMSC -HO-1. This is 
incorrectly stated at p10, l38 -42. 
- MDA levels are discussed, however, there are no results presented on MDA levels.  
- Again, what is the added value of BMSC -HO-1 over BMSC alone, according to cytokine 
measur ements? It seems not correctly described on p11, l9 -11. 
- Data on HO -1 should be discussed in more detail, since it is already known that HO -1 has 
protective effects in intestinal IR. What is the added value of BMSC -HO-1 over HO -1? 
- It should also be disc ussed what these findings could mean for patients suffering from intestinal 
IR. Can such therapies be translated to the human situation?  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary  controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any stati stics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five year s received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in a ny way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding , or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you  can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competi ng interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Acces s Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: The HO -1-expressing bone mesenchymal stem cells protects intestine from ischemia and  
reperfusion injury  
 
Version: 2 Date: 04 Dec 2018  
 
Reviewer: Kaatje Lenaerts  
 
Reviewer's report:  
 
General:  
- The manuscript describes a study on the protective potential of HO -1-expressing BMSC cells 
on the development of intestinal IR injury and survival in mice. Data are clearly presented; the 
experiments seem to be performed adequately. A clear protective of  the therapy is shown; 
however the novelty is limited since both BMSC cells and HO -1 has been shown to be protective 
in earlier studies. The effect of the combination is not clearly different from the effect of BMSC 
alone.  
- Incorrect use of scientific Eng lish throughout the manuscript text. Authors should ask a native 
English speaker from the biomedical field to revise the manuscript text.  
- Grammar mistakes are present throughout manuscript text.  
 
 
Background.  
- Intestinal IR is an important disease entit y with a high mortality but stating that "Ischemia 
reperfusion injury (I/R) is one of the major causes of high morbidity, disability, and mortality in 
the world." is too strong.  
- Background section is not focused, and a lot of duplicate information is pre sent.  
 
 
 
 
 
Materials & Methods:  
- Not clear why male SD rats are used in the study. As source of BMSCs? Seems not as they 
were purchased from a company.  
- Which primers were used? Refer to table where the sequences are shown.  
- Include power calculation to ju stify the number of animals per group used.  
- How where mice kept stable in during intestinal ischemia? E.g. was their temperature 
monitored? Was fluid administered? How was the abdomen closed? Etc.  
- Which intervention was performed in the sham group? Did  mice receive also PBS instead of 
BMSC?  
- Were 5 mice per group used for survival analysis and 5 mice for analyses after 24h? Hence, in 
n=10 in total per group? Or where 80 animals used instead of 40?  
- Which parameters were monitored during survival? Wher e animal welfare sheets used? Provide 
details on the health status of animals, and not only survival data.  
- Who performed the analysis of small intestinal injury? It is stated that it was performed by 
Lenaerts et al [16]. But reference 16 is an article by  Ceulemans et al. It could be that the authors 
mean that it was performed according to the article by Ceulemans et al. This should be correctly 
indicated.  
- Information on IR injury scoring is lacking. How many fields were scored at which 
magnification etc .? Was the scoring performed blindly?  
- Which statistical test was used to find significant differences? In the manuscript, only the post 
hoc test is mentioned.  
 
 
 
 
 
 
 
 
Results  
- Authors should explain the term MOI, p8, l1  
- Figure 1A. information on how many sam ples that were analyzed is missing.  
- Not clear why nuclear expression of GFP is higher in de BMSC/HO -1 group. What does this 
indicate?  
- It appears from Figure 1C a twofold increase was observed. This seems not a dramatic increase, 
as is stated on p8 l12/ 13 
- Correct labels should be used in all figures, since it is not clear what model and vector stands 
for. Use the terms sham - IR - BMSC - BMSC/HO -1 correctly. Also, in the manuscript text and 
legends this should be used consistently.  
- Again, increases in HO expression (Figure 3) are not dramatic.  
- Information on the plasma levels of the cytokines is missing. It is important to assess the 
potential of BMSCs on decreasing these systemic cytokines, as they play a large role in 
morbidity  of IR injury.  
- Information on the localization of the BMSCs in the injured tissue is lacking. As they are GFP -
positive, they can be tracked which would add clinically relevant information to the manuscript. 
Moreover, there phenotype could be assessed, to  evaluate their plasticity.  
- Are differences between Vector and HO -1 not significant in figure 4? What is the added value 
of HO -1? Also, in survival there were no differences between BMSC and BMSC -HO-1. 
 
Discussion  
- Survival rates were not significantly different between BMSC and BMSC -HO-1. This is 
incorrectly stated at p10, l38 -42. 
- MDA levels are discussed, however, there are no results presented on MDA levels.  
- Again, what is the added value of BMSC -HO-1 over BMSC alone, according to cytokine 
measur ements? It seems not correctly described on p11, l9 -11. 
- Data on HO -1 should be discussed in more detail, since it is already known that HO -1 has 
protective effects in intestinal IR. What is the added value of BMSC -HO-1 over HO -1? 
- It should also be disc ussed what these findings could mean for patients suffering from intestinal 
IR. Can such therapies be translated to the human situation?  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary  controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any stati stics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five year s received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in a ny way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding , or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you  can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competi ng interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Acces s Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant