Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes
Round 1
Reviewer 1 Report
In this manuscript, the authors established a method that hESCs/hiPSCs can be spontaneously differentiated into classical brown adipocytes (BA) in the absence of a cytokine cocktail, while only with the controlling of spheroids radiuses.I havea few questions about this manuscript.
1. Although this is a modified method for BAs induction, it will be much better, if the authors add some significant of BAs transplantation in Introduction.
2. In results 3.1.2, the authors used the immunostaining to demonstrate a BA morphology of the differentiated cells with abundant mitochondria and multilocular lipid droplets, however, the high power images may be needed. And the dead cells inside the size-controlled spheroid also need to be evaluated by immunostaining. Additionally, the homogeneity of the spheroid need to be evaluated by BAs’ specific markers staining or single cell sequence, to confirm the spheroid contained only one kind of cell (BAs). On the other hand, the passage ability of the induced BAs is also suggested to be confirmed.
3. In results 3.1.3, the authors showed a higher the dermal temperature of transplanted mice after several minutes from an administration of isoproterenol and intend to indicate that BAs produced from human pluripotent stem cells under cytokine-free conditions are functionally maturated. However, the results were only obtained from the mice after 48h transplantation, a result from a long term (one week or even four weeks) after transplantation is also needed to support the functionally maturation of transplanted BAs: 1) the authors need to confirm the transplanted BAs have the ability to survival in vivo, as the induced BAs were only survival in a size controlled manner, we don’t know whether they will survival after cells expansion in vivo, and the morphology will also need to be confirmed after long term transplantation; 2) the BAs are nerved by nervous systems, especially when response to stress, therefore, the evaluation of the cold response for the BAs mice after a long term transplantation, will give a more persuade support for BAs functionally maturation.
Author Response
Responses to the comments by Reviewer 1:
We truly appreciate valuable comments and suggestions.
According to the comments and suggestions, we added the following descriptions in our revised manuscript. We believe that our manuscript has been brushed up by responding to the comments/suggestions by reviewers.
Comment 1:
Although this is a modified method for BAs induction, it will be much better, if the authors add some significant of BAs transplantation in Introduction.
Response to the comment 1:
We added descriptions regarding that point in our revised manuscript in Introduction (lines 44- 52).
Comment 2:
In results 3.1.2, the authors used the immunostaining to demonstrate a BA morphology of the differentiated cells with abundant mitochondria and multilocular lipid droplets, however, the high power images may be needed. And the dead cells inside the size-controlled spheroid also need to be evaluated by immunostaining. Additionally, the homogeneity of the spheroid need to be evaluated by BAs’ specific markers staining or single cell sequence, to confirm the spheroid contained only one kind of cell (BAs). On the other hand, the passage ability of the induced BAs is also suggested to be confirmed.
Response to the comment 2:
In our revised manuscript, we added high power images of the differentiated cells (Figure 1c), the result of TUNEL assays to assess the existence of apoptotic cells in the spheroids (Figure 4) and the result of immunostaining study of the differentiated cells using an anti-UCP1 antibody (Figure 2d) as well as lipid droplet staining of the differentiated GAPDH-2A-NLS-TdTomato knock-in hESC (Figure 3) to show the quality of differentiation.
We also added descriptions regarding subculture-incompetence of BAs generated by our upgraded method as in the case of the BAs produced by our original method in Discussion (lines 613-616).
Comment 3:
In results 3.1.3, the authors showed a higher the dermal temperature of transplanted mice after several minutes from an administration of isoproterenol and intend to indicate that BAs produced from human pluripotent stem cells under cytokine-free conditions are functionally maturated. However, the results were only obtained from the mice after 48h transplantation, a result from a long term (one week or even four weeks) after transplantation is also needed to support the functionally maturation of transplanted BAs: 1) the authors need to confirm the transplanted BAs have the ability to survival in vivo, as the induced BAs were only survival in a size controlled manner, we don’t know whether they will survival after cells expansion in vivo, and the morphology will also need to be confirmed after long term transplantation; 2) the BAs are nerved by nervous systems, especially when response to stress, therefore, the evaluation of the cold response for the BAs mice after a long term transplantation, will give a more persuade support for BAs functionally maturation.
Response to the comment 3:
In our revised manuscript, we added the data regarding longer-term effects of transplanted BA along with the results to show the usefulness of cold acclimation to maintain biological activity of transplanted BA in Figure 10.
Reviewer 2 Report
The authors have done a very good study on how to differentiate iPS cells into brown adipocytes without using exogenous cytokines. The study is well done and manuscript is written well. I have few comments:
In line 56 authors should give approx. weight of human body. In other words, authors should state 150gm/--- kgs of human (average human body weight for this will suffice). Alternatively, mouse weight can be used (might be easier).
Is there difference of BAT levels between male and females? This should be stated.
The introduction should include a few lines about the role of each cytokine in differentiation. A brief sentence about each should suffice. I realize some of the functions may be unknown and this can be stated where applicable.
A table comparing this method to their old method along with advantages and disadvantages will be useful to readers.
Author Response
Responses to the comments by Reviewer 2:
We truly appreciate valuable comments and suggestions.
According to the comments and suggestions, we added the following descriptions in our revised manuscript. We believe that our manuscript has been brushed up by responding to the comments/suggestions by reviewers.
Comment 1:
In line 56 authors should give approx. weight of human body. In other words, authors should state 150gm/--- kgs of human (average human body weight for this will suffice). Alternatively, mouse weight can be used (might be easier).
Response to the comment 1:
We described in the revised manuscript that the weight of BAT in a form of “tissue weight/body weight” in humans (150 g/80 Kg = 1.8 g/Kg) and mice (150 mg/30 g = 5 mg/g, which was reported by Clerte M et al., Am J Soc Echocardiogr 26: 1465-1473, 2013) in Introduction (lines 61- 64).
Comment 2:
Is there difference of BAT levels between male and females? This should be stated.
Response to the comment 2:
We described in the revised manuscript that there are no differences in BAT levels between male and females when fully activated under a cool environment 19˚C for two hours, which was reported by Saito M et al., Diabetes 58: 1526-1531, 2009, in Introduction (lines 56- 68).
Comment 3:
The introduction should include a few lines about the role of each cytokine in differentiation. A brief sentence about each should suffice. I realize some of the functions may be unknown and this can be stated where applicable.
Response to the comment 3:
We described in the revised manuscript that the role of cytokines in the differentiation (BMP4 is required for generation of spheroid per se; BMP7 is required for expansion of BA progenitor; IL6 is required for functional maturation of BA for metabolism regulation; other cytokines seem to be involved in at least multilocular lipid droplet formation as suggested in our previous paper, Nishio et al., Cell Metab 2012) in Introduction (lines 83 - 88).
Comment 4:
A table comparing this method to their old method along with advantages and disadvantages will be useful to readers.
Response to the comment 4:
We added a table that summarizes the comparison between the up-graded method and the previous method along with advantages and disadvantages as below.
Round 2
Reviewer 1 Report
I think the results, which are in high quality, are enough to support the conclusion of this paper.
