Reviewer's report
Title:Instructive role of gut-derived factors on ES cells differentiation.
Version: 2Date:23 November 2010
Reviewer: Simon Kenny
Reviewer's report:
MAJOR COMPULSORY REVISIONS
Title: does not fully inform the reader of the subject matter of the paper which is
an evaluation of the utility ES cells as a potential source of cells for
transplantation
Background: I take issue with the misleading statement that Hirschsprungs
disease & pyloric stenosis results in a 'severe impairment of digestive functions,
for which conventional therapeutic approaches (surgical or pharmacological) are
mainly palliative and associated with poor quality of life for patients and
unacceptably high morbidity and mortality'. As a surgeon who does science I
have a responsibility to perform science at the same level as any scientist. I
would be grateful if scientists could return the favour. Firstly pyloric stenosis: the
mean length of stay for a child after surgery for pyloric stenosis is 48 hours -
children are discharged on full feeds and arent routinely followed up. The
complication rate in experienced hands is less than 2% and mortality is thankfully
a thing of the past. With regards to Hirschsprungs disease many children lead
perfectly normal lives with continence that matches their peers. Sadly about 20%
experience significant problems but modern surgical management means that
mortality is rare. This is not a trivial matter - when for example contemplating
stem cell based therapies the authors should fully appreciate that the risks
associated with these therapies may outweigh potential therapeutic benefits. It is
difficult to imagine that stem cell therapy will play a role in the treatment of pyloric
stenosis when surgery is cheap, safe and effective.
Characterization of neurospheres
The authors use the term neurospheres to describe the cell clusters derived by
culturing ES cells in non-adherent conditions in neurobasal medium. Expression
of nestin is considered enough to merit the neurosphere label but it would be
good to know what other markers are expressed at this stage. Are there p75
positive cells present? The Tuj1 neurons seen in Fig 2D-E look like neurons but a
1.8 fold increase is not massive. How does this level of expression compare with
neurospheres cultured from eg brain? The lack of expression of GFAP is also
worrying - did the authors check with RT-PCR?
RT_PCR Were the PCR products sequenced?
Coculture
This is potentially a very useful method and would shed light on the potential for
smooth muscle to influence ENS formation via humoral factors. It is
contaminated however by the presence of myenteric plexus - would it be possible
to exclude this either by using congenitally aganglionic bowel or by ablating
neuronal tissue? As it is there is potential for the neurons to suppress ES
neuronal differentiation
Intrapyloric transplantation
Was proliferation assessed in transplanted cells? This would be reassuring to
see. The authors quite rightly point out that longer time may be required for ES
derived cells to mature to the point that fully differentiated neurons are present in
the transplant (and in the coculture)
Figure 4 - It would be good to see PGP9.5 immuno alone as it doesn't look to
extend to neurites
Summary
This is a potentially interesting and well conceived paper that is let down at
present by poor characterization of putative ES derived neurospheres, the
inclusion of myenteric plexus neurons in the coculture experiment, and by not
following the behaviour of transplanted cells in vivo for a longer time. The title
needs revisiting and the final conclusion that ES-NS may not be suitable
candidates for ENS regeneration is in my view nihilistic - the only conclusion is
that the methods used by the authors have not worked but I do not believe that
they have exhausted this line of enquiry. Thank you for the opportunity to review
this potentially excellent paper.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
Reviewer's report
Title:Instructive role of gut-derived factors on ES cells differentiation.
Version: 2Date:23 November 2010
Reviewer: Simon Kenny
Reviewer's report:
MAJOR COMPULSORY REVISIONS
Title: does not fully inform the reader of the subject matter of the paper which is
an evaluation of the utility ES cells as a potential source of cells for
transplantation
Background: I take issue with the misleading statement that Hirschsprungs
disease & pyloric stenosis results in a 'severe impairment of digestive functions,
for which conventional therapeutic approaches (surgical or pharmacological) are
mainly palliative and associated with poor quality of life for patients and
unacceptably high morbidity and mortality'. As a surgeon who does science I
have a responsibility to perform science at the same level as any scientist. I
would be grateful if scientists could return the favour. Firstly pyloric stenosis: the
mean length of stay for a child after surgery for pyloric stenosis is 48 hours -
children are discharged on full feeds and arent routinely followed up. The
complication rate in experienced hands is less than 2% and mortality is thankfully
a thing of the past. With regards to Hirschsprungs disease many children lead
perfectly normal lives with continence that matches their peers. Sadly about 20%
experience significant problems but modern surgical management means that
mortality is rare. This is not a trivial matter - when for example contemplating
stem cell based therapies the authors should fully appreciate that the risks
associated with these therapies may outweigh potential therapeutic benefits. It is
difficult to imagine that stem cell therapy will play a role in the treatment of pyloric
stenosis when surgery is cheap, safe and effective.
Characterization of neurospheres
The authors use the term neurospheres to describe the cell clusters derived by
culturing ES cells in non-adherent conditions in neurobasal medium. Expression
of nestin is considered enough to merit the neurosphere label but it would be
good to know what other markers are expressed at this stage. Are there p75
positive cells present? The Tuj1 neurons seen in Fig 2D-E look like neurons but a
1.8 fold increase is not massive. How does this level of expression compare with
neurospheres cultured from eg brain? The lack of expression of GFAP is also
worrying - did the authors check with RT-PCR?
RT_PCR Were the PCR products sequenced?
Coculture
This is potentially a very useful method and would shed light on the potential for
smooth muscle to influence ENS formation via humoral factors. It is
contaminated however by the presence of myenteric plexus - would it be possible
to exclude this either by using congenitally aganglionic bowel or by ablating
neuronal tissue? As it is there is potential for the neurons to suppress ES
neuronal differentiation
Intrapyloric transplantation
Was proliferation assessed in transplanted cells? This would be reassuring to
see. The authors quite rightly point out that longer time may be required for ES
derived cells to mature to the point that fully differentiated neurons are present in
the transplant (and in the coculture)
Figure 4 - It would be good to see PGP9.5 immuno alone as it doesn't look to
extend to neurites
Summary
This is a potentially interesting and well conceived paper that is let down at
present by poor characterization of putative ES derived neurospheres, the
inclusion of myenteric plexus neurons in the coculture experiment, and by not
following the behaviour of transplanted cells in vivo for a longer time. The title
needs revisiting and the final conclusion that ES-NS may not be suitable
candidates for ENS regeneration is in my view nihilistic - the only conclusion is
that the methods used by the authors have not worked but I do not believe that
they have exhausted this line of enquiry. Thank you for the opportunity to review
this potentially excellent paper.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
Please review this paper.
user
Reviewer's report
Title:Instructive role of gut-derived factors on ES cells differentiation.
Version: 2Date:23 November 2010
Reviewer: Simon Kenny
Reviewer's report:
MAJOR COMPULSORY REVISIONS
Title: does not fully inform the reader of the subject matter of the paper which is
an evaluation of the utility ES cells as a potential source of cells for
transplantation
Background: I take issue with the misleading statement that Hirschsprungs
disease & pyloric stenosis results in a 'severe impairment of digestive functions,
for which conventional therapeutic approaches (surgical or pharmacological) are
mainly palliative and associated with poor quality of life for patients and
unacceptably high morbidity and mortality'. As a surgeon who does science I
have a responsibility to perform science at the same level as any scientist. I
would be grateful if scientists could return the favour. Firstly pyloric stenosis: the
mean length of stay for a child after surgery for pyloric stenosis is 48 hours -
children are discharged on full feeds and arent routinely followed up. The
complication rate in experienced hands is less than 2% and mortality is thankfully
a thing of the past. With regards to Hirschsprungs disease many children lead
perfectly normal lives with continence that matches their peers. Sadly about 20%
experience significant problems but modern surgical management means that
mortality is rare. This is not a trivial matter - when for example contemplating
stem cell based therapies the authors should fully appreciate that the risks
associated with these therapies may outweigh potential therapeutic benefits. It is
difficult to imagine that stem cell therapy will play a role in the treatment of pyloric
stenosis when surgery is cheap, safe and effective.
Characterization of neurospheres
The authors use the term neurospheres to describe the cell clusters derived by
culturing ES cells in non-adherent conditions in neurobasal medium. Expression
of nestin is considered enough to merit the neurosphere label but it would be
good to know what other markers are expressed at this stage. Are there p75
positive cells present? The Tuj1 neurons seen in Fig 2D-E look like neurons but a
1.8 fold increase is not massive. How does this level of expression compare with
neurospheres cultured from eg brain? The lack of expression of GFAP is also
worrying - did the authors check with RT-PCR?
RT_PCR Were the PCR products sequenced?
Coculture
This is potentially a very useful method and would shed light on the potential for
smooth muscle to influence ENS formation via humoral factors. It is
contaminated however by the presence of myenteric plexus - would it be possible
to exclude this either by using congenitally aganglionic bowel or by ablating
neuronal tissue? As it is there is potential for the neurons to suppress ES
neuronal differentiation
Intrapyloric transplantation
Was proliferation assessed in transplanted cells? This would be reassuring to
see. The authors quite rightly point out that longer time may be required for ES
derived cells to mature to the point that fully differentiated neurons are present in
the transplant (and in the coculture)
Figure 4 - It would be good to see PGP9.5 immuno alone as it doesn't look to
extend to neurites
Summary
This is a potentially interesting and well conceived paper that is let down at
present by poor characterization of putative ES derived neurospheres, the
inclusion of myenteric plexus neurons in the coculture experiment, and by not
following the behaviour of transplanted cells in vivo for a longer time. The title
needs revisiting and the final conclusion that ES-NS may not be suitable
candidates for ENS regeneration is in my view nihilistic - the only conclusion is
that the methods used by the authors have not worked but I do not believe that
they have exhausted this line of enquiry. Thank you for the opportunity to review
this potentially excellent paper.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
assistant