Reviewer's report
Title:Instructive role of gut-derived factors on ES cells differentiation.
Version: 2Date:23 November 2010
Reviewer: maria giuliana Giuliana vannucchi
Reviewer's report:
General comments
The manuscript entitled 'Instructive role of gut-derived factors on ES cells
differentiation.' by Valentina Sasselli, Maria-Adelaide Micci, Kristen M Kahrig and
Pankaj Jay Pasrichais is organized in two experimental part: the first using an ‘in
vitro model’ the second one using an ‘in vivo model’.
The ‘in vitro model’ is quite convincing and the theoretical principles that local
factors produced by the cells present in the bowel wall could influence the
neighbour stem cells and hopefully, induce them to acquire the phenotypes of the
cells present in this region sounds reasonable.
However, there are some data (see the single points below) whose inconsistency
or contradiction lie, in my opinion, on the kind of stem cells chosen.
In relation to the ‘in vivo model’ although theoretically working, it failed
completely.
I do not think, as the authors hypothesise, that is a time problem, rather these
very ‘young and almost totipotent cells, did not ‘feel’ the local and very
differentiated environment. Indeed, their location in the most inner part of the
circular muscle layer and, most of all, in the submucosa, suggests that these
cells injected on the serosal side, simple across the wall enable to stop and
likely, if you wait for much time they will be disappeared. In agreement with this
fate is the observation that these cells did not express any particular markers.
Therefore, after the authors have clarify and correct the points listed below, I
would suggest, in the attempt to make this study very significant, to investigate
the ultrastructure of the ES-N and their features. In this way it will possible to
understand what really happened to these cell before and after the commitment
taking into account that at both stage they express similar markers although in
different entity. With this investigation it will be possible to clarify whether they
really form synapses, whether they are so differentiated as they seems looking at
their shape in the immunefluorescence pictures etc...
Specific comments
Abstract
The first sentence in the conclusions listed in the Abstract should be attenuated
since
- no secreted factors was identified or assayed in this study,
- eventually one neuronal phenotype (nNOS) and not more, was detected.
Moreover, it has to be remembered that the antibody used for WB recognises a
nNOS isoform present in all type of neurons, independently of their phenotype an
also labelled the SMC. No image is present on the labelling distribution of the
polyclonal nNOS used for the immunohistochemical experiment. Why did the
authors use two diverse antibodies which labelled different isoforms. To my
experience the monoclonal one works in the immunohistochemistry as well.
Methods
There are too many sub-chapters.
For example the second and the third (pag. 5) should be collected in a single one
titles ‘Induction of neurospheres from mouse ES cells’ also because the title of
the third sub chapter does not correspond the content.
What is the DAPI exactly: compare pag 8 (a mounting media) to pag 9 (a nuclear
counterstaining).
Results
Second chapter, pag. 11
The authors show that the ES undifferentiated cells already express nestin and,
most of all, betaIIItubulin which are considered markers for neuronal
differentiation. How do the authors explain it? It seems that these markers are
not so specific being expressed by very undifferentiated cells such as those
coming out from the inner cell mass. Therefore, their increase in committed cells
could be aspecific as well.
Looking at the figure 2 and its legend, are the authors sure that the bar in the
Image A has the same value as the others bars?
Pag 13
In the text (row 7 from the up) is quoted Fig4D-I but no images corresponding to
these letters are contained in the Fig 4. Besides any reference to these letters is
missed in the related legend. The authors should look and make order.
Looking at the image of Fig.4 it seems that those corresponding to A-B are taken
earlier than those corresponding to C-D. It is true? Besides, in my experience to
exclude the presence of cell contacts (white arrows in 4C) only looking at these
image is an hazard unless you use specific antibody against specialized
structures or you look the cells at the TEM.
Finally, I was very surprised that the co-cultured cells did not express the glial
marker GFAP. It has been reported that in the myenteric neurosphere both,
neurons and glia cells differentiate when contacting the bowel wall and herein
organize in ganglia. The authors try to explain it in the discussion however, I still
wonder whether this model, with so undifferentiated cells, is really working!
In the text (row 8) the authors report a ‘more developed morphology’ of the
neurons labelled with alfaIII tubulin and they cite Fig.5A-D to show it. Indeed, Fig.
5 contains images of peripherin labelling, (5AandC) and alfaIII tubulin (5BandD).
Also here they have to be more precise.
Moreover, in this regard, I have a theoretical question: How could the authors get
immunohistochemical images of peripherin labelling in the control cells when, in
these cells, peripherin was almost undetectable by using WB? (see Fig.4B)
In regard to the alfa SMC labelling everything is very confused.
The authors affirm that the expression of this marker, although low, is increased
in co-cultured cells when compared to control cells. However, looking at the
images in Fig.6 it seems exactly the opposite. Besides, still in this case, I have
doubts on the bar value. Indeed, it seems that image 6A is at lower magnification
compared to image 6B.
Discussion
The authors show a correct criticism in discussing their data, nevertheless there
are parts too speculative (see the last paragraph pag. 15 on the SMC), full of
unanswered questions that need further studies (see pag 16).
Level of interest: An article of importance in its field
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: maria giuliana Giuliana vannucchi
Reviewer's report:
General comments
The manuscript entitled 'Instructive role of gut-derived factors on ES cells
differentiation.' by Valentina Sasselli, Maria-Adelaide Micci, Kristen M Kahrig and
Pankaj Jay Pasrichais is organized in two experimental part: the first using an ‘in
vitro model’ the second one using an ‘in vivo model’.
The ‘in vitro model’ is quite convincing and the theoretical principles that local
factors produced by the cells present in the bowel wall could influence the
neighbour stem cells and hopefully, induce them to acquire the phenotypes of the
cells present in this region sounds reasonable.
However, there are some data (see the single points below) whose inconsistency
or contradiction lie, in my opinion, on the kind of stem cells chosen.
In relation to the ‘in vivo model’ although theoretically working, it failed
completely.
I do not think, as the authors hypothesise, that is a time problem, rather these
very ‘young and almost totipotent cells, did not ‘feel’ the local and very
differentiated environment. Indeed, their location in the most inner part of the
circular muscle layer and, most of all, in the submucosa, suggests that these
cells injected on the serosal side, simple across the wall enable to stop and
likely, if you wait for much time they will be disappeared. In agreement with this
fate is the observation that these cells did not express any particular markers.
Therefore, after the authors have clarify and correct the points listed below, I
would suggest, in the attempt to make this study very significant, to investigate
the ultrastructure of the ES-N and their features. In this way it will possible to
understand what really happened to these cell before and after the commitment
taking into account that at both stage they express similar markers although in
different entity. With this investigation it will be possible to clarify whether they
really form synapses, whether they are so differentiated as they seems looking at
their shape in the immunefluorescence pictures etc...
Specific comments
Abstract
The first sentence in the conclusions listed in the Abstract should be attenuated
since
- no secreted factors was identified or assayed in this study,
- eventually one neuronal phenotype (nNOS) and not more, was detected.
Moreover, it has to be remembered that the antibody used for WB recognises a
nNOS isoform present in all type of neurons, independently of their phenotype an
also labelled the SMC. No image is present on the labelling distribution of the
polyclonal nNOS used for the immunohistochemical experiment. Why did the
authors use two diverse antibodies which labelled different isoforms. To my
experience the monoclonal one works in the immunohistochemistry as well.
Methods
There are too many sub-chapters.
For example the second and the third (pag. 5) should be collected in a single one
titles ‘Induction of neurospheres from mouse ES cells’ also because the title of
the third sub chapter does not correspond the content.
What is the DAPI exactly: compare pag 8 (a mounting media) to pag 9 (a nuclear
counterstaining).
Results
Second chapter, pag. 11
The authors show that the ES undifferentiated cells already express nestin and,
most of all, betaIIItubulin which are considered markers for neuronal
differentiation. How do the authors explain it? It seems that these markers are
not so specific being expressed by very undifferentiated cells such as those
coming out from the inner cell mass. Therefore, their increase in committed cells
could be aspecific as well.
Looking at the figure 2 and its legend, are the authors sure that the bar in the
Image A has the same value as the others bars?
Pag 13
In the text (row 7 from the up) is quoted Fig4D-I but no images corresponding to
these letters are contained in the Fig 4. Besides any reference to these letters is
missed in the related legend. The authors should look and make order.
Looking at the image of Fig.4 it seems that those corresponding to A-B are taken
earlier than those corresponding to C-D. It is true? Besides, in my experience to
exclude the presence of cell contacts (white arrows in 4C) only looking at these
image is an hazard unless you use specific antibody against specialized
structures or you look the cells at the TEM.
Finally, I was very surprised that the co-cultured cells did not express the glial
marker GFAP. It has been reported that in the myenteric neurosphere both,
neurons and glia cells differentiate when contacting the bowel wall and herein
organize in ganglia. The authors try to explain it in the discussion however, I still
wonder whether this model, with so undifferentiated cells, is really working!
In the text (row 8) the authors report a ‘more developed morphology’ of the
neurons labelled with alfaIII tubulin and they cite Fig.5A-D to show it. Indeed, Fig.
5 contains images of peripherin labelling, (5AandC) and alfaIII tubulin (5BandD).
Also here they have to be more precise.
Moreover, in this regard, I have a theoretical question: How could the authors get
immunohistochemical images of peripherin labelling in the control cells when, in
these cells, peripherin was almost undetectable by using WB? (see Fig.4B)
In regard to the alfa SMC labelling everything is very confused.
The authors affirm that the expression of this marker, although low, is increased
in co-cultured cells when compared to control cells. However, looking at the
images in Fig.6 it seems exactly the opposite. Besides, still in this case, I have
doubts on the bar value. Indeed, it seems that image 6A is at lower magnification
compared to image 6B.
Discussion
The authors show a correct criticism in discussing their data, nevertheless there
are parts too speculative (see the last paragraph pag. 15 on the SMC), full of
unanswered questions that need further studies (see pag 16).
Level of interest: An article of importance in its field
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: maria giuliana Giuliana vannucchi
Reviewer's report:
General comments
The manuscript entitled 'Instructive role of gut-derived factors on ES cells
differentiation.' by Valentina Sasselli, Maria-Adelaide Micci, Kristen M Kahrig and
Pankaj Jay Pasrichais is organized in two experimental part: the first using an ‘in
vitro model’ the second one using an ‘in vivo model’.
The ‘in vitro model’ is quite convincing and the theoretical principles that local
factors produced by the cells present in the bowel wall could influence the
neighbour stem cells and hopefully, induce them to acquire the phenotypes of the
cells present in this region sounds reasonable.
However, there are some data (see the single points below) whose inconsistency
or contradiction lie, in my opinion, on the kind of stem cells chosen.
In relation to the ‘in vivo model’ although theoretically working, it failed
completely.
I do not think, as the authors hypothesise, that is a time problem, rather these
very ‘young and almost totipotent cells, did not ‘feel’ the local and very
differentiated environment. Indeed, their location in the most inner part of the
circular muscle layer and, most of all, in the submucosa, suggests that these
cells injected on the serosal side, simple across the wall enable to stop and
likely, if you wait for much time they will be disappeared. In agreement with this
fate is the observation that these cells did not express any particular markers.
Therefore, after the authors have clarify and correct the points listed below, I
would suggest, in the attempt to make this study very significant, to investigate
the ultrastructure of the ES-N and their features. In this way it will possible to
understand what really happened to these cell before and after the commitment
taking into account that at both stage they express similar markers although in
different entity. With this investigation it will be possible to clarify whether they
really form synapses, whether they are so differentiated as they seems looking at
their shape in the immunefluorescence pictures etc...
Specific comments
Abstract
The first sentence in the conclusions listed in the Abstract should be attenuated
since
- no secreted factors was identified or assayed in this study,
- eventually one neuronal phenotype (nNOS) and not more, was detected.
Moreover, it has to be remembered that the antibody used for WB recognises a
nNOS isoform present in all type of neurons, independently of their phenotype an
also labelled the SMC. No image is present on the labelling distribution of the
polyclonal nNOS used for the immunohistochemical experiment. Why did the
authors use two diverse antibodies which labelled different isoforms. To my
experience the monoclonal one works in the immunohistochemistry as well.
Methods
There are too many sub-chapters.
For example the second and the third (pag. 5) should be collected in a single one
titles ‘Induction of neurospheres from mouse ES cells’ also because the title of
the third sub chapter does not correspond the content.
What is the DAPI exactly: compare pag 8 (a mounting media) to pag 9 (a nuclear
counterstaining).
Results
Second chapter, pag. 11
The authors show that the ES undifferentiated cells already express nestin and,
most of all, betaIIItubulin which are considered markers for neuronal
differentiation. How do the authors explain it? It seems that these markers are
not so specific being expressed by very undifferentiated cells such as those
coming out from the inner cell mass. Therefore, their increase in committed cells
could be aspecific as well.
Looking at the figure 2 and its legend, are the authors sure that the bar in the
Image A has the same value as the others bars?
Pag 13
In the text (row 7 from the up) is quoted Fig4D-I but no images corresponding to
these letters are contained in the Fig 4. Besides any reference to these letters is
missed in the related legend. The authors should look and make order.
Looking at the image of Fig.4 it seems that those corresponding to A-B are taken
earlier than those corresponding to C-D. It is true? Besides, in my experience to
exclude the presence of cell contacts (white arrows in 4C) only looking at these
image is an hazard unless you use specific antibody against specialized
structures or you look the cells at the TEM.
Finally, I was very surprised that the co-cultured cells did not express the glial
marker GFAP. It has been reported that in the myenteric neurosphere both,
neurons and glia cells differentiate when contacting the bowel wall and herein
organize in ganglia. The authors try to explain it in the discussion however, I still
wonder whether this model, with so undifferentiated cells, is really working!
In the text (row 8) the authors report a ‘more developed morphology’ of the
neurons labelled with alfaIII tubulin and they cite Fig.5A-D to show it. Indeed, Fig.
5 contains images of peripherin labelling, (5AandC) and alfaIII tubulin (5BandD).
Also here they have to be more precise.
Moreover, in this regard, I have a theoretical question: How could the authors get
immunohistochemical images of peripherin labelling in the control cells when, in
these cells, peripherin was almost undetectable by using WB? (see Fig.4B)
In regard to the alfa SMC labelling everything is very confused.
The authors affirm that the expression of this marker, although low, is increased
in co-cultured cells when compared to control cells. However, looking at the
images in Fig.6 it seems exactly the opposite. Besides, still in this case, I have
doubts on the bar value. Indeed, it seems that image 6A is at lower magnification
compared to image 6B.
Discussion
The authors show a correct criticism in discussing their data, nevertheless there
are parts too speculative (see the last paragraph pag. 15 on the SMC), full of
unanswered questions that need further studies (see pag 16).
Level of interest: An article of importance in its field
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