Reviewer ’s report  
 
Title: Helicobacter -induced Gastric Inflammation Alters the Properties of Gastric Tissue  
Stem/Progenitor Cells  
 
Version: 0 Date: 12 Sep 2017  
 
Reviewer: Guifang Xu  
 
Reviewer's report:  
 
This article by Shibata et al want to clarify the changes in epithelial stem/progenitor cells after 
chronic inflammation. This is an interesting research. The three -dimensional gastric epithelial 
cell culture system has been utilized . However, the precise mechanism has not been clarified. In 
this study only explained some phenomenons by mRNA expression and IHC analysis. According 
to current results, I think the authors can not come to the conclusions.  
 
Major  
1. The 3D organoid culture was from mice either infected , treated with MNU, or untreated 
control after 4 days  of culture,then, organoids were injected subcutaneously into mice and 
the  6  tumorigenicity and histological changes were analyzed after 2 months (page 7 line 3 -
7). Only the 4 days culture is enough to form organoid? We know the 3D organoid culture 
from the tumor tissue need long time can do the further research.  
2. In the methods about" 3 -dimentional organoid culture of gastric epithelial cells", the mice 
were killed , the  mouse gastric corpus was removed and cut into approximately 1 -mm2 
pieces. The  gastric epithelial  tissues  were washed only by PBS, during the procedure, any 
antibiotics has not been used, how to avoid the cell contamination? In the previous study of 
prostate cancer,  primocin was used during the cell culture[Unno K et al. Oncotarget. 2017 
Apr 19;8(31):51264 -51276. ].  
3. This study only examined the stem cell markers such as Cd44, Dclk1...,only the mRNA 
expression and IHC analysis have been analyzed. Just like the author explained the gastric 
cancer stem cells marker is not very specific. So I think the following conclusion "chronic 
inflammation induced by Helicobacter infection increased the number of tissue 
stem/progenitor cells, which acquired stemness properties, and altered the direction of 
differentiation toward intestinal metaplasia to cancer." cannot  been drown (page 3 line 7 -9). 
4. Figure 2D should select a picture which can show the whole area, not the regional part. I 
think the MUC4 expression tr end may be clear and more convincing.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comm ents to the authors.  
 
Yes 
 
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.  
 
Yes 
 
Are you able to assess any statistics 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:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing in terests, considering the following questions:  
 
1. Have you in the past five years 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 t he future?  
 
2. Do you hold any stocks or shares in an organisation that may in any 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 th e 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 h ave no competing 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 Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wis h 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: Helicobacter -induced Gastric Inflammation Alters the Properties of Gastric Tissue  
Stem/Progenitor Cells  
 
Version: 0 Date: 12 Sep 2017  
 
Reviewer: Guifang Xu  
 
Reviewer's report:  
 
This article by Shibata et al want to clarify the changes in epithelial stem/progenitor cells after 
chronic inflammation. This is an interesting research. The three -dimensional gastric epithelial 
cell culture system has been utilized . However, the precise mechanism has not been clarified. In 
this study only explained some phenomenons by mRNA expression and IHC analysis. According 
to current results, I think the authors can not come to the conclusions.  
 
Major  
1. The 3D organoid culture was from mice either infected , treated with MNU, or untreated 
control after 4 days  of culture,then, organoids were injected subcutaneously into mice and 
the  6  tumorigenicity and histological changes were analyzed after 2 months (page 7 line 3 -
7). Only the 4 days culture is enough to form organoid? We know the 3D organoid culture 
from the tumor tissue need long time can do the further research.  
2. In the methods about" 3 -dimentional organoid culture of gastric epithelial cells", the mice 
were killed , the  mouse gastric corpus was removed and cut into approximately 1 -mm2 
pieces. The  gastric epithelial  tissues  were washed only by PBS, during the procedure, any 
antibiotics has not been used, how to avoid the cell contamination? In the previous study of 
prostate cancer,  primocin was used during the cell culture[Unno K et al. Oncotarget. 2017 
Apr 19;8(31):51264 -51276. ].  
3. This study only examined the stem cell markers such as Cd44, Dclk1...,only the mRNA 
expression and IHC analysis have been analyzed. Just like the author explained the gastric 
cancer stem cells marker is not very specific. So I think the following conclusion "chronic 
inflammation induced by Helicobacter infection increased the number of tissue 
stem/progenitor cells, which acquired stemness properties, and altered the direction of 
differentiation toward intestinal metaplasia to cancer." cannot  been drown (page 3 line 7 -9). 
4. Figure 2D should select a picture which can show the whole area, not the regional part. I 
think the MUC4 expression tr end may be clear and more convincing.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comm ents to the authors.  
 
Yes 
 
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.  
 
Yes 
 
Are you able to assess any statistics 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:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing in terests, considering the following questions:  
 
1. Have you in the past five years 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 t he future?  
 
2. Do you hold any stocks or shares in an organisation that may in any 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 th e 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 h ave no competing 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 Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wis h 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: Helicobacter -induced Gastric Inflammation Alters the Properties of Gastric Tissue  
Stem/Progenitor Cells  
 
Version: 0 Date: 12 Sep 2017  
 
Reviewer: Guifang Xu  
 
Reviewer's report:  
 
This article by Shibata et al want to clarify the changes in epithelial stem/progenitor cells after 
chronic inflammation. This is an interesting research. The three -dimensional gastric epithelial 
cell culture system has been utilized . However, the precise mechanism has not been clarified. In 
this study only explained some phenomenons by mRNA expression and IHC analysis. According 
to current results, I think the authors can not come to the conclusions.  
 
Major  
1. The 3D organoid culture was from mice either infected , treated with MNU, or untreated 
control after 4 days  of culture,then, organoids were injected subcutaneously into mice and 
the  6  tumorigenicity and histological changes were analyzed after 2 months (page 7 line 3 -
7). Only the 4 days culture is enough to form organoid? We know the 3D organoid culture 
from the tumor tissue need long time can do the further research.  
2. In the methods about" 3 -dimentional organoid culture of gastric epithelial cells", the mice 
were killed , the  mouse gastric corpus was removed and cut into approximately 1 -mm2 
pieces. The  gastric epithelial  tissues  were washed only by PBS, during the procedure, any 
antibiotics has not been used, how to avoid the cell contamination? In the previous study of 
prostate cancer,  primocin was used during the cell culture[Unno K et al. Oncotarget. 2017 
Apr 19;8(31):51264 -51276. ].  
3. This study only examined the stem cell markers such as Cd44, Dclk1...,only the mRNA 
expression and IHC analysis have been analyzed. Just like the author explained the gastric 
cancer stem cells marker is not very specific. So I think the following conclusion "chronic 
inflammation induced by Helicobacter infection increased the number of tissue 
stem/progenitor cells, which acquired stemness properties, and altered the direction of 
differentiation toward intestinal metaplasia to cancer." cannot  been drown (page 3 line 7 -9). 
4. Figure 2D should select a picture which can show the whole area, not the regional part. I 
think the MUC4 expression tr end may be clear and more convincing.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comm ents to the authors.  
 
Yes 
 
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.  
 
Yes 
 
Are you able to assess any statistics 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:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing in terests, considering the following questions:  
 
1. Have you in the past five years 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 t he future?  
 
2. Do you hold any stocks or shares in an organisation that may in any 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 th e 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 h ave no competing 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 Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wis h 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