Reviewer's report
Title:Evaluation of ES-derived neural progenitors as a potential source for cell
replacement therapy in the gut.
Version: 4Date:20 July 2011
Reviewer: Robert O. Heuckeroth
Reviewer's report:
This is a revised manuscript that presents interesting and novel observations
about the effect of co-culture of ES cells with LM-MP preparations on neuronal
differentiation. The contrast between the ability of ES derived cells to adopt a
neural fate in vitro, improvement with LM-MP co-culture, and the lack of this type
of differentiation in vivo is worth reporting.
I am disappointed that the authors were unable to address either of the major
issues I raised in the original review, but certainly understand that limitations in
funding and personnel are practical considerations that cannot be easily
overcome. The paper “as is” raises enough interesting questions and
demonstrates a new co-culture model system that makes it interesting.
Major compulsory revisions: None.
Minor essential revisions: I found a number of additional minor issues with
grammar and data presentation that are detailed below and should ideally be
addressed. Sorry if I missed these on the first round.
1. First line on page 10: Why is “investigator” capitalized?
2. Figure legend # 1 is now quite confusing. There are two different descriptions
of 1A and 1B. Images go from A-J, but legend only goes from A-H. The
references to this figure in the Results section are equally confusing. For
example, on page 11 the reference to Figure 1C in Results discusses nestin
expression, but the image is phase contrast and not immunohistochemistry.
Authors should carefully review all of the figures, figure legends and references
in the text to be sure they match in this revised manuscript.
3. Page 11 reference to Figure 1D says that nestin was limited to a few cells
initially but increased significantly after neural induction. I see what I assume to
be nestin expression in Figure 1D, but there is no image for comparison showing
“few ES cells” expressing nestin. This problem may reflect the issues raised in
comment 2, but I think image 1C is not really what the authors intended to show.
4. Page 12: LMMP is used initially, but then authors use “LM-MP”. Please be
consistent.
5. Figure legend 4: The sentence beginning with “Pan neuronal” is confusing as
written. Please reread and revise for clarity and simplicity. I had to read 4 times.
Also the last sentence on page 25: “Neurite” refers to long thin extensions from
neurons (i.e. axon or dendrite) and is the correct word for this sentence. The
word “Neuritis” means inflammation of a neurite and should not be used in this
context. There is also inconsistency in nomenclature/abbreviations. Is an ES-NS
the same as an ES-NSC, the term used in the last sentence of this figure legend,
but not earlier in the legend? Why call CNS derived cells CNS-NSC and ES
derived cells ES-NS?
6. Page 14: The sentence “We also excluded that cell proliferation could partially
account for the increased levels of neuronal markers showed by analyzing . . .” is
very hard to read. It should say “We also demonstrated that increased cell
proliferation is unlikely to account for elevated levels of neuronal markers since
the percentage of Ki67 expressing cells is similar in the co-culture and control
groups.” It is OK to use alternative wording, but please revise the grammar for
simplicity and clarity.
7. Figure 6 D legend: The text here is confusing. It says that the qRT-PCR could
not be corroborated, but the Western blot image appears to correlate well with
the mRNA analysis, despite the inability to detect alpha SMA in control samples.
8. In many places, the legends for figures are so small that they are unlikely to be
legible when the images are at their final size in the journal. For example, the text
in Figure 1E, 2C, 4A-C, 5E, and 6C and D, should be much larger.
9. Figure 7D. What is demonstrated in this image? Presumably the red labeling is
DiI and simply shows that the transplanted cells survived, but here the red cells
are labeled SM indicating they are in the submucosal layer. In contrast, image in
A show some red cells in the muscle layer. Do the cells that end up in the muscle
layer express alpha-SMA? One would not expect transplanted cells that are in
the submucosa to express alpha-SMA (or at least they might be less likely to do
so). Can Image D be replaced with a comparable image that shows that
transplanted cells that end up in muscle are SMA negative?
Discretionary Revisions: None
Level of interest: An article of importance in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare I have no competing interests.
Reviewer's report
Title:Evaluation of ES-derived neural progenitors as a potential source for cell
replacement therapy in the gut.
Version: 4Date:20 July 2011
Reviewer: Robert O. Heuckeroth
Reviewer's report:
This is a revised manuscript that presents interesting and novel observations
about the effect of co-culture of ES cells with LM-MP preparations on neuronal
differentiation. The contrast between the ability of ES derived cells to adopt a
neural fate in vitro, improvement with LM-MP co-culture, and the lack of this type
of differentiation in vivo is worth reporting.
I am disappointed that the authors were unable to address either of the major
issues I raised in the original review, but certainly understand that limitations in
funding and personnel are practical considerations that cannot be easily
overcome. The paper “as is” raises enough interesting questions and
demonstrates a new co-culture model system that makes it interesting.
Major compulsory revisions: None.
Minor essential revisions: I found a number of additional minor issues with
grammar and data presentation that are detailed below and should ideally be
addressed. Sorry if I missed these on the first round.
1. First line on page 10: Why is “investigator” capitalized?
2. Figure legend # 1 is now quite confusing. There are two different descriptions
of 1A and 1B. Images go from A-J, but legend only goes from A-H. The
references to this figure in the Results section are equally confusing. For
example, on page 11 the reference to Figure 1C in Results discusses nestin
expression, but the image is phase contrast and not immunohistochemistry.
Authors should carefully review all of the figures, figure legends and references
in the text to be sure they match in this revised manuscript.
3. Page 11 reference to Figure 1D says that nestin was limited to a few cells
initially but increased significantly after neural induction. I see what I assume to
be nestin expression in Figure 1D, but there is no image for comparison showing
“few ES cells” expressing nestin. This problem may reflect the issues raised in
comment 2, but I think image 1C is not really what the authors intended to show.
4. Page 12: LMMP is used initially, but then authors use “LM-MP”. Please be
consistent.
5. Figure legend 4: The sentence beginning with “Pan neuronal” is confusing as
written. Please reread and revise for clarity and simplicity. I had to read 4 times.
Also the last sentence on page 25: “Neurite” refers to long thin extensions from
neurons (i.e. axon or dendrite) and is the correct word for this sentence. The
word “Neuritis” means inflammation of a neurite and should not be used in this
context. There is also inconsistency in nomenclature/abbreviations. Is an ES-NS
the same as an ES-NSC, the term used in the last sentence of this figure legend,
but not earlier in the legend? Why call CNS derived cells CNS-NSC and ES
derived cells ES-NS?
6. Page 14: The sentence “We also excluded that cell proliferation could partially
account for the increased levels of neuronal markers showed by analyzing . . .” is
very hard to read. It should say “We also demonstrated that increased cell
proliferation is unlikely to account for elevated levels of neuronal markers since
the percentage of Ki67 expressing cells is similar in the co-culture and control
groups.” It is OK to use alternative wording, but please revise the grammar for
simplicity and clarity.
7. Figure 6 D legend: The text here is confusing. It says that the qRT-PCR could
not be corroborated, but the Western blot image appears to correlate well with
the mRNA analysis, despite the inability to detect alpha SMA in control samples.
8. In many places, the legends for figures are so small that they are unlikely to be
legible when the images are at their final size in the journal. For example, the text
in Figure 1E, 2C, 4A-C, 5E, and 6C and D, should be much larger.
9. Figure 7D. What is demonstrated in this image? Presumably the red labeling is
DiI and simply shows that the transplanted cells survived, but here the red cells
are labeled SM indicating they are in the submucosal layer. In contrast, image in
A show some red cells in the muscle layer. Do the cells that end up in the muscle
layer express alpha-SMA? One would not expect transplanted cells that are in
the submucosa to express alpha-SMA (or at least they might be less likely to do
so). Can Image D be replaced with a comparable image that shows that
transplanted cells that end up in muscle are SMA negative?
Discretionary Revisions: None
Level of interest: An article of importance in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Evaluation of ES-derived neural progenitors as a potential source for cell
replacement therapy in the gut.
Version: 4Date:20 July 2011
Reviewer: Robert O. Heuckeroth
Reviewer's report:
This is a revised manuscript that presents interesting and novel observations
about the effect of co-culture of ES cells with LM-MP preparations on neuronal
differentiation. The contrast between the ability of ES derived cells to adopt a
neural fate in vitro, improvement with LM-MP co-culture, and the lack of this type
of differentiation in vivo is worth reporting.
I am disappointed that the authors were unable to address either of the major
issues I raised in the original review, but certainly understand that limitations in
funding and personnel are practical considerations that cannot be easily
overcome. The paper “as is” raises enough interesting questions and
demonstrates a new co-culture model system that makes it interesting.
Major compulsory revisions: None.
Minor essential revisions: I found a number of additional minor issues with
grammar and data presentation that are detailed below and should ideally be
addressed. Sorry if I missed these on the first round.
1. First line on page 10: Why is “investigator” capitalized?
2. Figure legend # 1 is now quite confusing. There are two different descriptions
of 1A and 1B. Images go from A-J, but legend only goes from A-H. The
references to this figure in the Results section are equally confusing. For
example, on page 11 the reference to Figure 1C in Results discusses nestin
expression, but the image is phase contrast and not immunohistochemistry.
Authors should carefully review all of the figures, figure legends and references
in the text to be sure they match in this revised manuscript.
3. Page 11 reference to Figure 1D says that nestin was limited to a few cells
initially but increased significantly after neural induction. I see what I assume to
be nestin expression in Figure 1D, but there is no image for comparison showing
“few ES cells” expressing nestin. This problem may reflect the issues raised in
comment 2, but I think image 1C is not really what the authors intended to show.
4. Page 12: LMMP is used initially, but then authors use “LM-MP”. Please be
consistent.
5. Figure legend 4: The sentence beginning with “Pan neuronal” is confusing as
written. Please reread and revise for clarity and simplicity. I had to read 4 times.
Also the last sentence on page 25: “Neurite” refers to long thin extensions from
neurons (i.e. axon or dendrite) and is the correct word for this sentence. The
word “Neuritis” means inflammation of a neurite and should not be used in this
context. There is also inconsistency in nomenclature/abbreviations. Is an ES-NS
the same as an ES-NSC, the term used in the last sentence of this figure legend,
but not earlier in the legend? Why call CNS derived cells CNS-NSC and ES
derived cells ES-NS?
6. Page 14: The sentence “We also excluded that cell proliferation could partially
account for the increased levels of neuronal markers showed by analyzing . . .” is
very hard to read. It should say “We also demonstrated that increased cell
proliferation is unlikely to account for elevated levels of neuronal markers since
the percentage of Ki67 expressing cells is similar in the co-culture and control
groups.” It is OK to use alternative wording, but please revise the grammar for
simplicity and clarity.
7. Figure 6 D legend: The text here is confusing. It says that the qRT-PCR could
not be corroborated, but the Western blot image appears to correlate well with
the mRNA analysis, despite the inability to detect alpha SMA in control samples.
8. In many places, the legends for figures are so small that they are unlikely to be
legible when the images are at their final size in the journal. For example, the text
in Figure 1E, 2C, 4A-C, 5E, and 6C and D, should be much larger.
9. Figure 7D. What is demonstrated in this image? Presumably the red labeling is
DiI and simply shows that the transplanted cells survived, but here the red cells
are labeled SM indicating they are in the submucosal layer. In contrast, image in
A show some red cells in the muscle layer. Do the cells that end up in the muscle
layer express alpha-SMA? One would not expect transplanted cells that are in
the submucosa to express alpha-SMA (or at least they might be less likely to do
so). Can Image D be replaced with a comparable image that shows that
transplanted cells that end up in muscle are SMA negative?
Discretionary Revisions: None
Level of interest: An article of importance in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare I have no competing interests.
assistant