Letter to the Editor 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.
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 ckit-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 knockin 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 knockins 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: 1. 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. 2. 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
(Circ Res. 2014;115:e38-e39.) © 2014 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.115.305380
Downloaded from http://circres.ahajournals.org/ ate38 CONS CALIFORNIA DIG LIB on December 9, 2014
Nadal-Ginard et al Response to Letter to the Editor e39 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
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).
Disclosures None.
Bernardo Nadal-Ginard 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
Daniele Torella Molecular and Cellular Cardiology Department of Medical and Surgical Sciences Magna Graecia University Catanzaro, Italy
References 1. Nadal-Ginard B, Ellison GM, Torella D. Absence of evidence is not evidence of absence: pitfalls of cre knock-ins in the c-Kit locus. Circ Res. 2014;115:415–418. 2. Molkentin JD. Letter by Molkentin Regarding Article, “The Absence of Evidence Is Not Evidence of Absence: The Pitfalls of Cre Knock-Ins in the c-Kit Locus”. Circ Res. 2014;115:e21–e23. 3. van Berlo JH, Kanisicak O, Maillet M, Vagnozzi RJ, Karch J, Lin SC, Middleton RC, Marbán E, Molkentin JD. c-kit+ cells minimally contribute cardiomyocytes to the heart. Nature. 2014;509:337–341. 4. Ellison GM, Torella D, Dellegrottaglie S, et al Endogenous cardiac stem cell activation by insulin-like growth factor-1/hepatocyte growth factor intracoronary injection fosters survival and regeneration of the infarcted pig heart. J Am Coll Cardiol. 2011;58:977–986. 5. Ellison GM, Vicinanza C, Smith AJ, et al Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair. Cell. 2013;154:827–842. 6. Smith AJ, Lewis FC, Aquila I, Waring CD, Nocera A, Agosti V, NadalGinard B, Torella D, Ellison GM. Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart. Nat Protoc. 2014;9:1662–1681. 7. Schmidt-Supprian M, Rajewsky K. Vagaries of conditional gene targeting. Nat Immunol. 2007;8:665–668. 8. Smith L. Good planning and serendipity: exploiting the Cre/Lox system in the testis. Reproduction. 2011;141:151–161. 9. Zhou BO, Yue R, Murphy MM, Peyer JG, Morrison SJ. Leptin-receptorexpressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell. 2014;15:154–168. 10. Lennartsson J, Rönnstrand L. Stem cell factor receptor/c-Kit: from basic science to clinical implications. Physiol Rev. 2012;92:1619–1649. 11. Bernex F, De Sepulveda P, Kress C, Elbaz C, Delouis C, Panthier JJ. Spatial and temporal patterns of c-kit-expressing cells in WlacZ/+ and WlacZ/WlacZ mouse embryos. Development. 1996;122:3023–3033. 12. Berrozpe G, Agosti V, Tucker C, Blanpain C, Manova K, Besmer P. A distant upstream locus control region is critical for expression of the Kit receptor gene in mast cells. Mol Cell Biol. 2006;26:5850–5860. 13. Nadal-Ginard B, Ellison GM, Torella D. The cardiac stem cell compartment is indispensable for myocardial cell homeostasis, repair and regeneration in the adult. Stem Cell Res. 2014. doi: 10.1016/j.scr.2014.04.008. [Epub ahead of print]
Downloaded from http://circres.ahajournals.org/ at CONS CALIFORNIA DIG LIB on December 9, 2014
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'' Bernardo Nadal-Ginard, Georgina M. Ellison and Daniele Torella Circ Res. 2014;115:e38-e39 doi: 10.1161/CIRCRESAHA.115.305380 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/115/12/e38
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/
Downloaded from http://circres.ahajournals.org/ at CONS CALIFORNIA DIG LIB on December 9, 2014
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 ckit-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 knockin 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 knockins 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: 1. 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. 2. 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
(Circ Res. 2014;115:e38-e39.) © 2014 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.115.305380
Downloaded from http://circres.ahajournals.org/ ate38 CONS CALIFORNIA DIG LIB on December 9, 2014
Nadal-Ginard et al Response to Letter to the Editor e39 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
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).
Disclosures None.
Bernardo Nadal-Ginard 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
Daniele Torella Molecular and Cellular Cardiology Department of Medical and Surgical Sciences Magna Graecia University Catanzaro, Italy
References 1. Nadal-Ginard B, Ellison GM, Torella D. Absence of evidence is not evidence of absence: pitfalls of cre knock-ins in the c-Kit locus. Circ Res. 2014;115:415–418. 2. Molkentin JD. Letter by Molkentin Regarding Article, “The Absence of Evidence Is Not Evidence of Absence: The Pitfalls of Cre Knock-Ins in the c-Kit Locus”. Circ Res. 2014;115:e21–e23. 3. van Berlo JH, Kanisicak O, Maillet M, Vagnozzi RJ, Karch J, Lin SC, Middleton RC, Marbán E, Molkentin JD. c-kit+ cells minimally contribute cardiomyocytes to the heart. Nature. 2014;509:337–341. 4. Ellison GM, Torella D, Dellegrottaglie S, et al Endogenous cardiac stem cell activation by insulin-like growth factor-1/hepatocyte growth factor intracoronary injection fosters survival and regeneration of the infarcted pig heart. J Am Coll Cardiol. 2011;58:977–986. 5. Ellison GM, Vicinanza C, Smith AJ, et al Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair. Cell. 2013;154:827–842. 6. Smith AJ, Lewis FC, Aquila I, Waring CD, Nocera A, Agosti V, NadalGinard B, Torella D, Ellison GM. Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart. Nat Protoc. 2014;9:1662–1681. 7. Schmidt-Supprian M, Rajewsky K. Vagaries of conditional gene targeting. Nat Immunol. 2007;8:665–668. 8. Smith L. Good planning and serendipity: exploiting the Cre/Lox system in the testis. Reproduction. 2011;141:151–161. 9. Zhou BO, Yue R, Murphy MM, Peyer JG, Morrison SJ. Leptin-receptorexpressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell. 2014;15:154–168. 10. Lennartsson J, Rönnstrand L. Stem cell factor receptor/c-Kit: from basic science to clinical implications. Physiol Rev. 2012;92:1619–1649. 11. Bernex F, De Sepulveda P, Kress C, Elbaz C, Delouis C, Panthier JJ. Spatial and temporal patterns of c-kit-expressing cells in WlacZ/+ and WlacZ/WlacZ mouse embryos. Development. 1996;122:3023–3033. 12. Berrozpe G, Agosti V, Tucker C, Blanpain C, Manova K, Besmer P. A distant upstream locus control region is critical for expression of the Kit receptor gene in mast cells. Mol Cell Biol. 2006;26:5850–5860. 13. Nadal-Ginard B, Ellison GM, Torella D. The cardiac stem cell compartment is indispensable for myocardial cell homeostasis, repair and regeneration in the adult. Stem Cell Res. 2014. doi: 10.1016/j.scr.2014.04.008. [Epub ahead of print]
Downloaded from http://circres.ahajournals.org/ at CONS CALIFORNIA DIG LIB on December 9, 2014
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'' Bernardo Nadal-Ginard, Georgina M. Ellison and Daniele Torella Circ Res. 2014;115:e38-e39 doi: 10.1161/CIRCRESAHA.115.305380 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/115/12/e38
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/
Downloaded from http://circres.ahajournals.org/ at CONS CALIFORNIA DIG LIB on December 9, 2014
