Reader Comments

Post a new comment on this article

Referee comments: Referee 3

Posted by PLOS_ONE_Group on 17 Apr 2008 at 15:10 GMT

Referee 3's review:

**********
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.
**********

This study explores the source, morphology and cardiogenic potential of a population of small round refractile cells derived from mice cardiac explants and probably arising from circulating cells.

Although these cells shared some characteristics of cardiac myocytes in vitro and survived engraftment in the adult heart in vivo, they failed to differentiate into functional cardiac myocytes in vivo.

This study is based on the assumption that the cell population isolated from cardiac explants is comparable to that described in reference 1 and 3. However this assumption is wrong for the following reasons:

1. In ref 1 and 3 isolated myocardial tissue was washed and digested trice and only the remaining tissue fragments were washed and cultured as explants. This critical step was omitted in this study.

2. In any case the EDC, as well as the cells collected from the explants described by Messina et al) represent a mixed population. In the cited references (1, 3 of the manuscript), a pre-selection on poly-D-Lisine is performed allowing the attachment of the fibroblast-like cells and the growth in suspension of cellular spheres derived from single elements.

3. Culture media employed were different (both for the explants cultures than for the spheres).

4. In references 1 and 3, phase-bright cells were collected by mild trypsin digestion, under visual control at room temperature

5. Cells obtained in ref. 1 and 3 were clonogenic, expressed stem cells antigens (ckit), and differentiated into cardiac myocytes: spontaneously beating cardiospheres were obtained from mice (the clones derived from TnI-nLcZ and MLC3F-nLacZ transgenic mice became blue after histochemical staining and show a beating phenotype)

6. A similar kind of method to isolate stem cells has been employed by Sampaolesi et al (Nature. 2006 Nov 30;444(7119):574-9.). In that study, explants cultures obtained from muscle biopsy give rise to same kind of cells, identified as mesoangioblasts,.

Therefore all experiments should be repeated by using the correct method of cell isolation from explants in order to compare the results and draw reasonable conclusions.

The only interest of this study is that cells obtained from heart explants without previous digestion probably are blood derived and not cardiogenic.

RE: Referee comments: Referee 3

Galinanes replied to PLOS_ONE_Group on 30 Apr 2008 at 14:20 GMT

This was our response to the reviewer's comments, prior to publication. The manuscript was amended in light of these.

While we take note of the reviewer’s comments, we strongly disagree with them. From the comments, we felt that reviewer 3 has completely misconceived the aims and messages of this paper. The aim of this paper was not to prove or disprove that the EDCs we observed are the same as those in reference 1 & 3, nor to prove that those cells from these references are or are not cardiac progenitors as implied by the comments. Hence, the comment: “This study is based on the assumption that the cell population isolated from cardiac explants is comparable to that described in reference 1 and 3” was wrong, as we merely described some of the shared similarities of our EDCs with those cells described in reference 1 & 3, despite some differences in culture conditions. What is important in our studies is that the EDCs obtained under the culture conditions used do not behave like cardiac progenitors using different lineage tracing techniques. It is also worth noticing the comment made by one of the reviewers – “the importance of rigorous characterization of any cell type that is projected to be used in a clinical setting”. Whether cells obtained using the “correct methods” as described by reviewer 3 can withstand similar rigorous characterization is not the aim of this study, but another scientific question & paper that needs to be addressed separately, till then it may be premature to conclude a method of cell isolation as “correct”. Furthermore, there were also flaws in the supporting statements used to substantiate this reviewer’s comments such as:

1) “In ref 1 and 3 isolated myocardial tissue was washed and digested trice and only the remaining tissue fragments were washed and cultured as explants. This critical step was omitted in this study”: Whilst we washed the cardiac explants with PBS by vigorous washing by re-suspension we don’t believe this technique is efficacious enough to remove circulating cells that may remain in the capillaries in the center of the explant. The hearts needed to be perfused for up to 10 minutes in order not to see any red cells from the coronary sinus. In ref 1 and 3 the myocardial tissue was only washed (not perfused) and digested thrice, and then cultured as explants. The phase bright populations from refs 1 and 3 appeared after 2 weeks and were described as a mixed population. They shared similar morphological characteristics as the cells seen in our setup, despite slightly different isolation techniques. However, we have to re-emphasise that to compare & contrast our cells with those previously described was not the objective of our studies.

2) “In any case the EDC, as well as the cells collected from the explants described by Messina et al represent a mixed population. In the cited references (1, 3 of the manuscript), a pre-selection on poly-D-Lisine is performed allowing the attachment of the fibroblast-like cells and the growth in suspension of cellular spheres derived from single elements”: As described above, the aim of our studies was not to prove or disprove that the EDCs we observed are the same as those in reference 1 & 3, nor to prove that those cells from these references are or are not cardiac progenitors.

3) “Culture media employed were different (both for the explants cultures than for the spheres)”: Again we acknowledge that the culture conditions used in reference 1&3 and in the present studies were not identical.

4) “In references 1 and 3, phase-bright cells were collected by mild trypsin digestion, under visual control at room temperature”: Use of mild trypsin digestion to select for the EDCs used in reference 1 and 3 may not guarantee that the cloned that formed cardiospheres were necessarily derived from the small round cells because most cells round up into spheres after digestion and when cultured in suspension form spherical clusters.

5) “Cells obtained in ref. 1 and 3 were clonogenic, expressed stem cells antigens (ckit), and differentiated into cardiac myocytes: spontaneous beating cardiospheres were obtained from mice (the clones derived from TnI-nLcZ and MLC3F-nLacZ transgenic mice became blue after histochemical staining and show a beating phenotype)”: Once more, our objective was not to compare whether the EDCs obtained in the present studies were identical to the cells obtained in other studies. Ref 1 used neonatal and postnatal hearts where the cardiac myocytes can still undergo rounds of division and have migratory capacity. These explants have therefore long been used in migratory assays for neonatal and early post natal cardiac explants[1]. Thus cells from early postnatal explants may contain cardiac myocytes that have migrated from the heart having undergone rounds of cell division to appear as if they are cardiac progenitors in cardiospheres.

6) “A similar kind of method to isolate stem cells has been employed by Samaolesi et al (Nature 2006 Nov 30;444(7119):574-9.). In that study, explants cultures obtained from muscle biopsy give rise to same kind of cells, identified as mesoangioblasts”: Sampaolesi’s technique[2] to isolate mesangioblasts from muscle explants does not use enzyme digestion as implied by reviewer 3. Here embryonic tissue explants are cultured for a week after which the explants are dissociated and cells isolated.

[1] Bock-Marquette I, Saxena A, White MD, Dimaio JM, Srivastava D. Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair. Nature. 2004 Nov 25; 432(7016): 466-72.
[2] Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D'Antona G, Pellegrino MA, et al. Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science (New York, NY. 2003 Jul 25; 301(5632): 487-92.