Response to Molkentin’s Letter to The Editor Regarding Article, “The Absence of Evidence Is Not Evidence of Absence: The Pitfalls of Cre Knock-Ins in the c-kit Locus”
To the Editor:
In the article referred to1 in Jeffery D. Molkentin’s letter,2 we evaluated the article from his laboratory entitled c-kit+ Cells Minimally Contribute Cardiomyocytes to the Heart.3 We pointed out that the van Berlo et al’s article (hereafter van Berlo) has serious methodological weaknesses and lacks some crucial controls that together do not allow reaching many of their conclusions and certainly do not support the title.
Molkentin challenges most, if not all, of the scientific basis for our critiques, yet his letter does not provide any new experimental information or data, which was missing from the original article.3 van Berlo did not identify, characterize, or isolate a single bona fide c-kit+ cardiac stem/progenitor cell (abbreviated either as CSC or CPC). The statements in Molkentin’s letter2 and van Berlo3 about the analysis of CPCs are misleading. Whether “the lungs are green, the gastrointestinal tract was green, the bone marrow was green, but the heart was not”2 has no bearing on the issue under discussion because of the recombination in the lungs and other tissues is neither correlated nor a predictor of what happened to the CPCs; the van Berlo is focused on the fate of the cardiomyocyte precursors. The statement that “van Berlo, used fluorescence-activated cell sorter imaging cytometry to show that ≈80% of cardiac CPCs labeled by the c-kit antibody are recombined by the Cre approach and expressed…eGFP and well >80% in the bone marrow”2 is flawed because the authors did not isolate or identify CPCs or hematopoietic stem cells, a necessary condition to determine the degree of recombination. The imaging cytometry used in van Berlo3 gated the entire c-kit+ cell population in the myocardium and bone marrow. Contrary to the quote above, it has been repeatedly shown that only a small percentage (2%–8%) of c-kit+ cells in the heart or bone marrow are CPCs or hematopoietic stem cells, respectively. The overwhelming majority of c-kit+ cells in the heart are mast cells (CD45+/c-kit+/tryptase+) and endothelial progenitors/precursors (CD34+/CD31+/c-kit+), which are several-fold higher in number than the CPCs.4–6 These data have been nicely corroborated by van Berlo.3 van Berlo3 and Molkentin’s letter2 also disregard that among the c-kit+ cells of wild-type animals, the CPCs and hematopoietic stem cells express c-kit at a significantly lower level than the c-kit+ mast cells and endothelial progenitor cells.4–7 They did notice3 that in c-kit-mutated animals (constitutive and regulated), Cre expression depends on only 1 of the alleles. Therefore, it is to be expected that c-kit (and therefore Cre) expression would be only 50% in the already low expressing c-kit+/CD45–/CD34–/CD31–/Tryptase CPCs.4–6 Whether this is enough to allow recombination in CPCs is the central issue left unanswered by van Berlo.3 Without this information, we will continue to maintain that the conclusions in van Berlo are not supported by the data presented and that the role of the c-kit+ CPCs in adult cardiac homeostasis and repair is as we documented in our Cell article.5
The Cre-lox system, particularly when combined with the knock-in of Cre (constitutive or regulatable) in a particular genetic locus of interest, has proven to be valuable for cell fate tracking in development and in the adult.7 Unfortunately, however, it is not unerring and there are several cases where the data obtained with Cre-lox knock-ins has been proven to be incorrect.8,9 To track the fate of a given cell type with the Cre-lox system where Cre is driven by a particular cell-specific promoter in a knock-in which abolishes the expression of the natural gene product of the mutated allele (a null allele), as is the case of van Berlo,3 certain parameters need to be carefully monitored in the homozyguous animal. This is more important when the homozygous mutant phenotype is lethal in the fetal or early perinatal period, as is the case for the c-kit knock-ins reported by van Berlo.3 Before engaging in complex experiments and reaching conclusions, particularly when these conclusions challenge accepted science, the experimental system used must fulfill ≥2 conditions:
It is indispensable to ascertain experimentally that the approach used is able to provide valid answers to the questions posed by the experimentalist. This is particularly important when the level of expression of the knocked-in gene in the cells to be tracked is low or very low.9 More so if its function is essential to generate the cell fate phenotype, that is the subject of the study. This is so because tracking the fate of these cells is completely dependent on the production of sufficient Cre by the mutated allele to produce the recombination of the marker gene (the floxed reporter) in each cell of the cohort whose fate is to be tracked or in a fraction of them large enough to be representative of the entire cohort. If this condition is not met, the knocked-in animals will not be able to track the fate of any cells expressing Cre below the level needed to produce recombination of the floxed allele in the same cell.
When the protein product of the locus knocked-in has an essential role in producing the phenotype used for the readout of the fate of the tracked cell and this function is exquisitely sensitive to the quantitative expression of the gene, the effect of producing a null gene on the phenotype of the homozyguous animal needs to be fully explored before embarking in the fate-tracking exercise. Again, this is the case for the c-kit gene, which is expressed and indispensable for the proliferation, commitment, and differentiation of most, if not all, multipotent embryonic and adult stem cell types. The importance of the quantitative levels of expression and function of c-kit has been documented in multiple publications describing the different phenotypes of the mutants of this locus—the White (W) locus.10
Neither of these 2 controls together with the identification and isolation of the c-kit+ CSCs/CPCs, which we think to be indispensable to track the fate of the CSCs/CPCs, were performed by the Molkentin group.3
Finally, contrary to Molkentin’s statements, to date there is no reported c-kit transgenic or knock-in mouse that faithfully recapitulates the developmental and postnatal expression of the endogenous c-kit gene.11,12 Also, there is no publication in which any one of us has said, either as author or as a coauthor, that “expression levels of c-kit in CSCs/CPCs are similar to bone marrow cells.”2 What we and others have repeatedly documented is that the CSCs/CPCs express c-kit and are negative for CD45, CD34, CD31, and tryptase, and we have shown fluorographs and images, which, without exception, show a relatively low level of c-kit expression in CSCs/CPCs.
In conclusion, whether the role of the cardiac c-kit+ CSCs/CPCs is the one reported by us repeatedly13 and in particular in Ellison et al,5 or, on the contrary, is as reported by van Berlo3 is an important issue for cardiovascular medicine. The answer will likely have great effect on the future of myocardial repair/regenerative procedures. Addressing the shortcomings of van Berlo’s experiments using the same mouse strain for complete consistency is what needs to be done. Thus, to resolve this controversy in the accepted scientific manner, we propose to repeat the experiments reported by van Berlo,3 and we are open to everyone willing to repeat those reported by us in Ellison et al.5
Georgina M. Ellison
Center of Human and Aerospace Physiological Sciences and
Center for Stem Cells and Regenerative Medicine
Faculty of Medicine & Life Sciences
King’s College London
London, United Kingdom
Molecular and Cellular Cardiology
Department of Medical and Surgical Sciences
Magna Graecia University
Sources of Funding
Drs Nadal-Ginard and Ellison are supported by EU FP7 large scale collaborative project grants CARE-MI (Health-F5-2010-242038) and Endostem (Health F5-2010-241440). Dr Torella is supported by FIRB-Futuro-in-Ricerca (RBFR081CCS, RBFR12I3KA), and Italian Ministry of Health (GR-2008-1142673, GR-2010-2318945).
Letters to the Editor will be published, if suitable, as space permits. They should not exceed 1000 words (typed double-spaced) in length and may be subject to editing or abridgment.
- © 2014 American Heart Association, Inc.
- Nadal-Ginard B,
- Ellison GM,
- Torella D
- Molkentin JD
- Smith L
- Lennartsson J,
- Rönnstrand L
- Bernex F,
- De Sepulveda P,
- Kress C,
- Elbaz C,
- Delouis C,
- Panthier JJ
- Berrozpe G,
- Agosti V,
- Tucker C,
- Blanpain C,
- Manova K,
- Besmer P
- Nadal-Ginard B,
- Ellison GM,
- Torella D