Reviewer's report
Title:Instructive role of gut-derived factors on ES cells differentiation.
Version: 2Date:19 November 2010
Reviewer: Robert O. Heuckeroth
Reviewer's report:
This is a well written and interesting manuscript that fills a gap in the emerging
and exciting literature about the use of various types of stem cells to generate
enteric neurons and glia. The authors use a divided chamber to demonstrate that
secreted factors from gut smooth muscle and myenteric plexus layers can
increase neuronal differentiation of ES derived neurospheres in culture. They
also demonstrate surprisingly that the same ES-NS do not appear to differentiate
into neurons when transplanted into the pyloric muscle in vivo. While it is not
surprising that gut derived factors influence ES-NS differentiation, I’m surprised
there is enough released into the media to have an effect. The in vivo/in vitro
difference is also striking and interesting. In addition, the authors are correct that
this assay system opens an opportunity to identify critical factors affecting
differentiation. The discussion is balanced and the points they make are well
supported by data.
Major compulsory revisions:
1. Page 11: It would be great if they could provide cell numbers in addition to
Western blot quantitative data to support the discussion in Figure 2. Authors say
they generate significantly more neurons with their protocol. How many more?
Increased tubulin data are great, but could reflect higher levels of expression in
differentiated cells instead of more cells that differentiate. Similarly, are there
more nestin + cells or simply higher levels of nestin in the same number of cells?
This is important for understanding the results.
2. When I reached the end of the Results section, I “wanted a bit more” reason to
hope that ES-NS might have therapeutic utility in vivo. It was disappointing that
these cells do not appear to differentiate into neurons in vivo, even though the
result is interesting. It is difficult to know based on the data provided how to
overcome this barrier, but a couple of relatively easy additional experiments
might make the paper a lot better.
a. Would injecting ES-NS exposed to LM-MP be more likely to differentiate into
neurons or glia in vivo than ES-NS that had not been exposed to LM-MP? Given
what the rest of the paper is about, I actually expected to see this experiment
done when I got to the end of the results section.
b. As the authors suggest, perhaps waiting longer would permit neuronal
differentiation of ES-NS in vivo. I recognize that this is an open ended experiment
since it is not clear how long to wait, but a couple of experiments with a
considerably longer time frame (e.g. a month) would make the paper much
better.
c. If the ES-NS do not differentiate in vivo, they may continue to proliferate,
causing cancer. Experiments in “a” and “b” above would also provide insight into
how to overcome this problem.
To be clear: I do not think that the issues in # 2 absolutely need to be addressed,
but they would make the paper much “more satisfying” and may not be too
difficult for the authors to complete. I would particularly be interested in “2a”,
since if ES-NS exposed to LM-MP do not form neurons in vivo, the problems may
be even more complex than anticipated.
Minor essential revisions:
Title: Should it be “ES cells” or “ES cell”?
Page 2: Last line of the abstract should be “at” instead of “al”.
Page 3: Title word “Background” is not spelled correctly.
Page 3, last line of first paragraph: “In this prospect” probably should be “In this
respect” or another phrase. Meaning is unclear.
Page 3: “bioptic” should probably be replaced by the word “biopsy”. “Bioptic” is
used in a different context. The same sentence should begin: “Recent evidence
has” instead of “evidences have”.
Page 4: “GMEM (Sigma)” is a bit ambiguous. At the Sigma site there are three
different media including Glasgow’s minimal essential and Glasgow’s modified
Eagle’s medium. It is important to be clear about media components.
Page 5: Consider “externa” instead of “externae”. Both are probably correct, but
the first is more common and plural is not needed.
Page 9, line 6: “investigator who” instead of “Investigator that”.
Page 10: Animal studies committee approval information probably does not
belong under the section entitled “Statistical analysis”.
Page 14, 4th line from the bottom: “. . . LM-MP plays” is awkward and seems like
a remnant from an earlier edited version. How about “LM-MP has”?
Level of interest: An article of outstanding merit and interest in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
No competing interests.
Reviewer's report
Title:Instructive role of gut-derived factors on ES cells differentiation.
Version: 2Date:19 November 2010
Reviewer: Robert O. Heuckeroth
Reviewer's report:
This is a well written and interesting manuscript that fills a gap in the emerging
and exciting literature about the use of various types of stem cells to generate
enteric neurons and glia. The authors use a divided chamber to demonstrate that
secreted factors from gut smooth muscle and myenteric plexus layers can
increase neuronal differentiation of ES derived neurospheres in culture. They
also demonstrate surprisingly that the same ES-NS do not appear to differentiate
into neurons when transplanted into the pyloric muscle in vivo. While it is not
surprising that gut derived factors influence ES-NS differentiation, I’m surprised
there is enough released into the media to have an effect. The in vivo/in vitro
difference is also striking and interesting. In addition, the authors are correct that
this assay system opens an opportunity to identify critical factors affecting
differentiation. The discussion is balanced and the points they make are well
supported by data.
Major compulsory revisions:
1. Page 11: It would be great if they could provide cell numbers in addition to
Western blot quantitative data to support the discussion in Figure 2. Authors say
they generate significantly more neurons with their protocol. How many more?
Increased tubulin data are great, but could reflect higher levels of expression in
differentiated cells instead of more cells that differentiate. Similarly, are there
more nestin + cells or simply higher levels of nestin in the same number of cells?
This is important for understanding the results.
2. When I reached the end of the Results section, I “wanted a bit more” reason to
hope that ES-NS might have therapeutic utility in vivo. It was disappointing that
these cells do not appear to differentiate into neurons in vivo, even though the
result is interesting. It is difficult to know based on the data provided how to
overcome this barrier, but a couple of relatively easy additional experiments
might make the paper a lot better.
a. Would injecting ES-NS exposed to LM-MP be more likely to differentiate into
neurons or glia in vivo than ES-NS that had not been exposed to LM-MP? Given
what the rest of the paper is about, I actually expected to see this experiment
done when I got to the end of the results section.
b. As the authors suggest, perhaps waiting longer would permit neuronal
differentiation of ES-NS in vivo. I recognize that this is an open ended experiment
since it is not clear how long to wait, but a couple of experiments with a
considerably longer time frame (e.g. a month) would make the paper much
better.
c. If the ES-NS do not differentiate in vivo, they may continue to proliferate,
causing cancer. Experiments in “a” and “b” above would also provide insight into
how to overcome this problem.
To be clear: I do not think that the issues in # 2 absolutely need to be addressed,
but they would make the paper much “more satisfying” and may not be too
difficult for the authors to complete. I would particularly be interested in “2a”,
since if ES-NS exposed to LM-MP do not form neurons in vivo, the problems may
be even more complex than anticipated.
Minor essential revisions:
Title: Should it be “ES cells” or “ES cell”?
Page 2: Last line of the abstract should be “at” instead of “al”.
Page 3: Title word “Background” is not spelled correctly.
Page 3, last line of first paragraph: “In this prospect” probably should be “In this
respect” or another phrase. Meaning is unclear.
Page 3: “bioptic” should probably be replaced by the word “biopsy”. “Bioptic” is
used in a different context. The same sentence should begin: “Recent evidence
has” instead of “evidences have”.
Page 4: “GMEM (Sigma)” is a bit ambiguous. At the Sigma site there are three
different media including Glasgow’s minimal essential and Glasgow’s modified
Eagle’s medium. It is important to be clear about media components.
Page 5: Consider “externa” instead of “externae”. Both are probably correct, but
the first is more common and plural is not needed.
Page 9, line 6: “investigator who” instead of “Investigator that”.
Page 10: Animal studies committee approval information probably does not
belong under the section entitled “Statistical analysis”.
Page 14, 4th line from the bottom: “. . . LM-MP plays” is awkward and seems like
a remnant from an earlier edited version. How about “LM-MP has”?
Level of interest: An article of outstanding merit and interest in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
No competing interests.
Please review this paper.
user
Reviewer's report
Title:Instructive role of gut-derived factors on ES cells differentiation.
Version: 2Date:19 November 2010
Reviewer: Robert O. Heuckeroth
Reviewer's report:
This is a well written and interesting manuscript that fills a gap in the emerging
and exciting literature about the use of various types of stem cells to generate
enteric neurons and glia. The authors use a divided chamber to demonstrate that
secreted factors from gut smooth muscle and myenteric plexus layers can
increase neuronal differentiation of ES derived neurospheres in culture. They
also demonstrate surprisingly that the same ES-NS do not appear to differentiate
into neurons when transplanted into the pyloric muscle in vivo. While it is not
surprising that gut derived factors influence ES-NS differentiation, I’m surprised
there is enough released into the media to have an effect. The in vivo/in vitro
difference is also striking and interesting. In addition, the authors are correct that
this assay system opens an opportunity to identify critical factors affecting
differentiation. The discussion is balanced and the points they make are well
supported by data.
Major compulsory revisions:
1. Page 11: It would be great if they could provide cell numbers in addition to
Western blot quantitative data to support the discussion in Figure 2. Authors say
they generate significantly more neurons with their protocol. How many more?
Increased tubulin data are great, but could reflect higher levels of expression in
differentiated cells instead of more cells that differentiate. Similarly, are there
more nestin + cells or simply higher levels of nestin in the same number of cells?
This is important for understanding the results.
2. When I reached the end of the Results section, I “wanted a bit more” reason to
hope that ES-NS might have therapeutic utility in vivo. It was disappointing that
these cells do not appear to differentiate into neurons in vivo, even though the
result is interesting. It is difficult to know based on the data provided how to
overcome this barrier, but a couple of relatively easy additional experiments
might make the paper a lot better.
a. Would injecting ES-NS exposed to LM-MP be more likely to differentiate into
neurons or glia in vivo than ES-NS that had not been exposed to LM-MP? Given
what the rest of the paper is about, I actually expected to see this experiment
done when I got to the end of the results section.
b. As the authors suggest, perhaps waiting longer would permit neuronal
differentiation of ES-NS in vivo. I recognize that this is an open ended experiment
since it is not clear how long to wait, but a couple of experiments with a
considerably longer time frame (e.g. a month) would make the paper much
better.
c. If the ES-NS do not differentiate in vivo, they may continue to proliferate,
causing cancer. Experiments in “a” and “b” above would also provide insight into
how to overcome this problem.
To be clear: I do not think that the issues in # 2 absolutely need to be addressed,
but they would make the paper much “more satisfying” and may not be too
difficult for the authors to complete. I would particularly be interested in “2a”,
since if ES-NS exposed to LM-MP do not form neurons in vivo, the problems may
be even more complex than anticipated.
Minor essential revisions:
Title: Should it be “ES cells” or “ES cell”?
Page 2: Last line of the abstract should be “at” instead of “al”.
Page 3: Title word “Background” is not spelled correctly.
Page 3, last line of first paragraph: “In this prospect” probably should be “In this
respect” or another phrase. Meaning is unclear.
Page 3: “bioptic” should probably be replaced by the word “biopsy”. “Bioptic” is
used in a different context. The same sentence should begin: “Recent evidence
has” instead of “evidences have”.
Page 4: “GMEM (Sigma)” is a bit ambiguous. At the Sigma site there are three
different media including Glasgow’s minimal essential and Glasgow’s modified
Eagle’s medium. It is important to be clear about media components.
Page 5: Consider “externa” instead of “externae”. Both are probably correct, but
the first is more common and plural is not needed.
Page 9, line 6: “investigator who” instead of “Investigator that”.
Page 10: Animal studies committee approval information probably does not
belong under the section entitled “Statistical analysis”.
Page 14, 4th line from the bottom: “. . . LM-MP plays” is awkward and seems like
a remnant from an earlier edited version. How about “LM-MP has”?
Level of interest: An article of outstanding merit and interest in its field
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
No competing interests.
assistant