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closeReferee comments: Referee 1 (Kenneth R Chien)
Posted by PLOS_ONE_Group on 17 Apr 2008 at 15:08 GMT
Referee 1's review (Kenneth R Chien):
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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.
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The results are important, albeit it negative, as they reflect on cell- based therapy for heart disease that may be entering the clinic and emphasizes the importance of rigorous characterization of any cell type that is projected to be used in a clinical setting. The studies also emphasize the need for functional evaluation of the differentiated progeny following transplantation, not just relying on molecular markers, and also suggests that the cells that have been cloned out of adult heart tissue may be hematopoietic in original and have properties similar to tissue specific macrophages. TEH lineage tracing approach that is utilized is state of the art, and distinguishes this work from much of the work that has been done and published by others, reinforcing the importance of lineage tracing as a gold standard to track cell lineage fate after transplantation.
Taken together the study is an important negative result that has many important points related to the best means to evaluate potential and putative cardiac progenitors in the future. There are a couple of points that should be addressed noted below:
1) A recent study notes that there are c-Kit positive cells that are blood derived in many organs, including heart, that are resident hematopoietic cells that serve in immunosurveilllance. This work was just published and these cells could be similar to the cells reported in this and other studies that have attempted to coax clonal derived cells from heart explants. The study should be referenced and ad it would be of interest to determine if the two cells types are similar or not.
Massberg S, Schaerli P, Knezevic-Maramica I, Köllnberger M, Tubo N, Moseman
EA, Huff IV, Junt T, Wagers AJ, Mazo IB, von Andrian UH.
Immunosurveillance by hematopoietic progenitor cells trafficking through blood,
lymph, and peripheral tissues.
Cell. 2007 Nov 30;131(5):994-1008.
2) It would be nice to provide a transcriptional profile on the cells in the current study so as to potentially provide a point of comparison with the cells isolated from other groups. How similar are the culture conditions to those reported by the other groups?
3) One convincing way to show that these cells are blood derived is to perform a bone marrow transplantation study with tagged cells. This would enhance the results of the study by unequivocally showing the hematopoietic origin of the tissue specific cells that are clonally derived from this type of explant strategy.
RE: Referee comments: Referee 1 (Kenneth R Chien)
Galinanes replied to PLOS_ONE_Group on 30 Apr 2008 at 14:15 GMT
This was our response to the reviewer's comments, prior to publication. Our manuscript was amended to incorprate these changes:
1) c-Kit positive cells that are blood derived reside in many organs including the heart: Thanks again to this reviewer for attracting our attention to the important paper by Massberg et al. Certainly, in the revised version we have discussed our results in the context of the findings reported by this paper and, as suggested, the paper has been incorporated to the references.
2) Transcriptional profile: We agree with the reviewer that a transcriptional profile will be important for future studies. In the present studies we have performed RT-PCR for cardiac markers, and observed that the transcription factor GATA-4 was expressed by the EDCs. Despite this, EDCs failed to attain a full cardiac phenotype in vitro and in vivo. It is uncertain what the expression of a specific transcriptional profile may mean for the final fate of cells; for example, other investigators have shown that the expression of stem cell transcription factors such as OCT-4 are not synonymous with pluripotency or embryonic stem cell properties. Regarding the culture conditions between our studies and previous studies, we recognise that there were some differences but we still observed similar morphological characteristics as pointed out in the revised manuscript.
3) Mice with chimeric bone marrow: As suggested by the reviewer, culturing explants from mice with chimeric tagged bone marrow cells may represent a good technique of delineating whether the EDCs are of bone marrow lineage or whether they depend on the stimulation of circulating blood derived factors. However, whilst the use of mice chimeric bone marrow would further confirm the extracardiac origin of EDCs, it will not modify the major findings of the manuscript in that the EDCs regardless of their origin do not exhibit the potential to differentiate into functional cardiac myocytes under the present culture conditions. Therefore the suggested experiment will not affect the interpretation of our results and the impact of the manuscript. We fully appreciate the importance of the suggested experiment and the question to be answered but feel that it will better addressed in a separate manuscript.