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. published online June 25, 2014; 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/early/2014/06/26/CIRCRESAHA.114.304676
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 University of Texas--Austin on June 28, 2014
COMMENTARIES ON CUTTING EDGE SCIENCE
The Absence of Evidence Is Not Evidence of Absence: The Pitfalls of Cre Knock-Ins in the C-Kit Locus Bernardo Nadal-Ginard1, Georgina M. Ellison1,2, Daniele Torella2 1
Centre of Human and Aerospace Physiological Sciences & Centre for Stem Cells and Regenerative Medicine, School of Biomedical Sciences, King’s College, London, UK 2Molecular and Cellular Cardiology, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy. .
Commentary on: c-kit1 cells minimally contribute cardiomyocytes to the heart van Berlo et al Nature. 2014. doi:10.1038/nature13309
Address for correspondence: Bernardo Nadal-Ginard, MD, PhD Centre of Human and Aerospace Physiological Sciences & Centre for Stem Cells and Regenerative Medicine School of Biomedical Sciences King's College London Shepherd's House, Rm 4.16 Guy's Campus London SE1 1UL e-mail: [email protected]
Daniele Torella, MD, PhD Molecular and Cellular Cardiology Dept of Medical and Surgical Sciences Magna Graecia University Campus S. Venuta, Viale Europa 88100, Catanzaro, Italy e-mail: [email protected]
The opinions expressed in this Commentary are not necessarily those of the editors or of the American Heart Association. Commentaries serve as a forum in which experts highlight and discuss articles (published here and elsewhere) that the editors of Circulation Research feel are of particular significance to cardiovascular medicine. Commentaries are edited by Aruni Bhatnagar & Ali J. Marian.
DOI: 10.1161/CIRCRESAHA.114.304676 Downloaded from http://circres.ahajournals.org/ at University of Texas--Austin on June 28, 2014
1
A recent paper by Jeffrey Molkentin’s group using mice with Cre/lox knock-in in the c-kit locus concludes that, contrary to other reports, c-kit+ cells minimally contribute cardiomyocytes to the heart. We believe the authors’ interpretation of the results is problematic because critical data to support their conclusions have not been provided. Recently, van Berlo et al. (1) published a paper in Nature entitled “c-kit+ cells minimally contribute cardiomyocytes to the heart”, in which they assessed the contribution of c-kit expressing cell lineages to the formation of cardiomyocytes and microvasculature in the myocardium during development, ageing and following myocardial infarction (MI). The authors knocked-in the c-kit locus a constitutive Cre-IRES-nGFP in one line of mice and a tamoxifen-inducible mER-Cre-mER in another line. Both gene knock-ins placed the CRE sequence in frame with the ATG start codon in exon 1of the Kit gene. These mice were cross-bred with the ROSA26 reporter (R26R) mutated mice carrying a floxed (and Cre dependent) reporter gene (either GFP or mT/mG construct). Based on a limited set of experiments, the authors concluded that although cardiac c-kit+ cells contribute cardiomyocytes, they do so minimally (from ≈0.001% to ≈0.02%) and, therefore, it was considered to be functionally insignificant. By contrast, c-kit+ cells amply generated cardiac endothelial cells. From these data the authors concluded that the use of c-kit+ cells in myocardial repair and regeneration protocols should be reconsidered. Interestingly, this paper comes out at a particularly propitious time to challenge the regenerative properties of the c-kit+ cardiac progenitor cells. Over the past several months a flurry of negative reports in the scientific and lay press have predicted the demise of stem cell-based regenerative medicine in general and myocardial regeneration/repair in particular. While this new critical assessment might be justified for a field used to overblown and to uncritical claims about its potential, it has created confusion among the lay population at large and the research community in particular. Curiously, the results of van Berlo et al., directly contradict two papers published ten years apart (2,3) and many other papers in between by us and other groups. A variety of experimental approaches including cell fate tracking, genetic tagging, cell transplantation, etc. have shown that the adult heart harbors bona fide tissue specific endogenous resident cardiac stem cells (eCSCs) that among their surface receptors, express the stem cell factor tyrosine kinase receptor, c-kit. These cells are multipotent, clonogenic and self-renewing. The progeny of a single cell forms multipotent spheres (cardiospheres) and differentiate in vitro and in vivo into cardiomyocytes, vascular smooth muscle and endothelial cells. Furthermore, the adult myocardium is dependent on the regenerative capacity of the eCSCs to anatomically and functionally repair diffuse myocardial damage (3). If the eCSC response to this diffuse damage is ablated, the spontaneous endogenous heart repair is blunted with animals going into heart failure. When functional eCSCs are repopulated through exogenous administration of clonogenic eCSCs, anatomical damage and functional cardiac impairment are completely and permanently reverted. If these regenerated cells are selectively killed, the recovery is reversed and the animals return to heart failure with a significant increase in cardiac death. Taken together, these data demonstrate that c-kit expressing eCSCs are necessary and sufficient for myocardial regeneration and repair in the adult heart (3). In short, although both groups agree that myocardial c-kit+ cells can generate cardiomyocytes and vascular cells in the adult, the role assigned to the c-kit+ cells in adult cardiac homeostasis and repair is diametrically opposed. Although the conclusions of each group are supported by apparently solid experimental data, the conclusions reached are incompatible. Both cannot be correct. In science, positive results, unless their methodology and/or interpretation can be challenged, usually trump negative ones. These two conflicting reports, in addition to technical and methodological differences and questions arising about the generation of the data, also pose important questions about
DOI: 10.1161/CIRCRESAHA.114.304676 Downloaded from http://circres.ahajournals.org/ at University of Texas--Austin on June 28, 2014
2
myocardial biology and the prospects of developing effective regeneration/repair protocols: Is the adult myocardium a self-renewing tissue? If so, what is the physiological significance of the self-renewal? Are the c-kit+ cells the regenerative/repair agents? If not, which are these regenerative agents? Clarification of the reason(s) for the reported discrepancies is of high priority, not only for cardiac biology but also for the future of the field of myocardial repair and regeneration. Discrepant and even contradictory results on a given topic are nothing new in science, even when the experiments are properly planned and the results accurately reported. In most cases the discrepancies are often due to a misinterpretation of the power of the experimental system(s) used which often leads to the over- and/or mis-interpretation of the results. The latter appears to be main issue with the van Berlo et al. paper. Not all Cre/LoxP cell-tracking systems provide reliable answers One perennial conundrum in cell biology has been the search for an efficient and accurate method of tracking the cell lineage origin as well as the fate of a given cell type, both during development and in adulthood. The development of the Cre/LoxP system of constitutive and conditional gene targeting together with trans- or knocked-in genes seemed the ideal procedure for fate mapping of specific cells. Inducible or regulated Cre-expressing mouse lines are increasingly popular because they significantly improve temporal and spatial targeting of genes (4). The most widely used inducible Cre lines utilise a Cre fused to a mutant ligand-binding domain of the oestrogen receptor (Cre-ER, mER-Cre-mER or CreERT2) where Cre-ER, mER-Cre-mER or Cre-ERT2 activation depends on the selective oestrogen receptor modulator tamoxifen (4). Although the constitutive and conditional Cre/loxP systems are both extremely powerful, they has several drawbacks that need to be properly controlled to avoid erroneous interpretation of observed results (4). As the van Berlo et al., work is exclusively based on the efficiency and fidelity of the Cre knock-in in the c-kit locus to track c-kit+ cell lineage fate, one must consider the main shortcomings of this recombination system that are summarized in Table 1. Therefore, considering these shortcomings, the conclusions reached with the Cre/lox system are only as reliable as the specific controls carried out to insure that the system performance is appropriate to unambiguously answer the questions addressed by the experiment. Table 2 summarizes the three controls that are particularly important. Unless these conditions are fulfilled the results of a Cre/lox experiment are un-interpretable. This is even more so when the results are negative because there is low or absent identifiable marked progeny of the target cells, as is the case of the results of van Berlo et al. These caveats are particularly important in light of the very complex and yet not fully elucidated c-kit gene regulation (6). In fact, all the c-kit transgenic or knock-ins produced to track c-kit expressing cells are far from closely recapitulating c-kit expression and function either in development or adult life (6,7). Does the c-kit/Cre knock-in recombine the reporter gene in the c-kit+ eCSCs? It is evident that both the constitutive and regulated Cre c-kit gene mutated mice lines used by van Berlo et al., are c-kit hypomorphs, most likely hemizygous null, which are equivalent to the W/+ mutant mice (8). While the typical signs of a c-kit hemizygous null allele (white spots on the belly, paws and tip of the tail) are not clearly reported in the paper, this conclusion is confirmed by the fact that the homozygous animals are not viable at birth and mostly die in utero. Whether the level of Cre expression
DOI: 10.1161/CIRCRESAHA.114.304676 Downloaded from http://circres.ahajournals.org/ at University of Texas--Austin on June 28, 2014
3
from the mutated allele is sufficient to produce recombination in all or most of the c-kit expressing cells is unknown, except that there is recombination in cells/tissues with high endogenous c-kit expression. The authors provided data showing recombination in several cell types, including myocardial cells, but not in the cells relevant to the results, i.e. the c-kit+ eCSCs. The reported recombination in total c-kit+ bone marrow cells and in myocyte-depleted c-kit+ cardiac cells is not relevant to the efficacy of the Cre/lox set-up because it is not specified which are the c-kit+ cell types recombined and which are not. In the constitutive Cre-IRES-nGFPxR26R-eGFP mice if the recombination were spatially and temporally correct, the majority (if not all) of blood cells in the adult should be labelled, as c-kit is expressed in all the embryonic cells with hematopoietic activity. Yet this piece of evidence is not provided. Furthermore, bone marrow and myocardial c-kit+ cells are complex cell mixtures with
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/early/2014/06/26/CIRCRESAHA.114.304676
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 University of Texas--Austin on June 28, 2014
COMMENTARIES ON CUTTING EDGE SCIENCE
The Absence of Evidence Is Not Evidence of Absence: The Pitfalls of Cre Knock-Ins in the C-Kit Locus Bernardo Nadal-Ginard1, Georgina M. Ellison1,2, Daniele Torella2 1
Centre of Human and Aerospace Physiological Sciences & Centre for Stem Cells and Regenerative Medicine, School of Biomedical Sciences, King’s College, London, UK 2Molecular and Cellular Cardiology, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy. .
Commentary on: c-kit1 cells minimally contribute cardiomyocytes to the heart van Berlo et al Nature. 2014. doi:10.1038/nature13309
Address for correspondence: Bernardo Nadal-Ginard, MD, PhD Centre of Human and Aerospace Physiological Sciences & Centre for Stem Cells and Regenerative Medicine School of Biomedical Sciences King's College London Shepherd's House, Rm 4.16 Guy's Campus London SE1 1UL e-mail: [email protected]
Daniele Torella, MD, PhD Molecular and Cellular Cardiology Dept of Medical and Surgical Sciences Magna Graecia University Campus S. Venuta, Viale Europa 88100, Catanzaro, Italy e-mail: [email protected]
The opinions expressed in this Commentary are not necessarily those of the editors or of the American Heart Association. Commentaries serve as a forum in which experts highlight and discuss articles (published here and elsewhere) that the editors of Circulation Research feel are of particular significance to cardiovascular medicine. Commentaries are edited by Aruni Bhatnagar & Ali J. Marian.
DOI: 10.1161/CIRCRESAHA.114.304676 Downloaded from http://circres.ahajournals.org/ at University of Texas--Austin on June 28, 2014
1
A recent paper by Jeffrey Molkentin’s group using mice with Cre/lox knock-in in the c-kit locus concludes that, contrary to other reports, c-kit+ cells minimally contribute cardiomyocytes to the heart. We believe the authors’ interpretation of the results is problematic because critical data to support their conclusions have not been provided. Recently, van Berlo et al. (1) published a paper in Nature entitled “c-kit+ cells minimally contribute cardiomyocytes to the heart”, in which they assessed the contribution of c-kit expressing cell lineages to the formation of cardiomyocytes and microvasculature in the myocardium during development, ageing and following myocardial infarction (MI). The authors knocked-in the c-kit locus a constitutive Cre-IRES-nGFP in one line of mice and a tamoxifen-inducible mER-Cre-mER in another line. Both gene knock-ins placed the CRE sequence in frame with the ATG start codon in exon 1of the Kit gene. These mice were cross-bred with the ROSA26 reporter (R26R) mutated mice carrying a floxed (and Cre dependent) reporter gene (either GFP or mT/mG construct). Based on a limited set of experiments, the authors concluded that although cardiac c-kit+ cells contribute cardiomyocytes, they do so minimally (from ≈0.001% to ≈0.02%) and, therefore, it was considered to be functionally insignificant. By contrast, c-kit+ cells amply generated cardiac endothelial cells. From these data the authors concluded that the use of c-kit+ cells in myocardial repair and regeneration protocols should be reconsidered. Interestingly, this paper comes out at a particularly propitious time to challenge the regenerative properties of the c-kit+ cardiac progenitor cells. Over the past several months a flurry of negative reports in the scientific and lay press have predicted the demise of stem cell-based regenerative medicine in general and myocardial regeneration/repair in particular. While this new critical assessment might be justified for a field used to overblown and to uncritical claims about its potential, it has created confusion among the lay population at large and the research community in particular. Curiously, the results of van Berlo et al., directly contradict two papers published ten years apart (2,3) and many other papers in between by us and other groups. A variety of experimental approaches including cell fate tracking, genetic tagging, cell transplantation, etc. have shown that the adult heart harbors bona fide tissue specific endogenous resident cardiac stem cells (eCSCs) that among their surface receptors, express the stem cell factor tyrosine kinase receptor, c-kit. These cells are multipotent, clonogenic and self-renewing. The progeny of a single cell forms multipotent spheres (cardiospheres) and differentiate in vitro and in vivo into cardiomyocytes, vascular smooth muscle and endothelial cells. Furthermore, the adult myocardium is dependent on the regenerative capacity of the eCSCs to anatomically and functionally repair diffuse myocardial damage (3). If the eCSC response to this diffuse damage is ablated, the spontaneous endogenous heart repair is blunted with animals going into heart failure. When functional eCSCs are repopulated through exogenous administration of clonogenic eCSCs, anatomical damage and functional cardiac impairment are completely and permanently reverted. If these regenerated cells are selectively killed, the recovery is reversed and the animals return to heart failure with a significant increase in cardiac death. Taken together, these data demonstrate that c-kit expressing eCSCs are necessary and sufficient for myocardial regeneration and repair in the adult heart (3). In short, although both groups agree that myocardial c-kit+ cells can generate cardiomyocytes and vascular cells in the adult, the role assigned to the c-kit+ cells in adult cardiac homeostasis and repair is diametrically opposed. Although the conclusions of each group are supported by apparently solid experimental data, the conclusions reached are incompatible. Both cannot be correct. In science, positive results, unless their methodology and/or interpretation can be challenged, usually trump negative ones. These two conflicting reports, in addition to technical and methodological differences and questions arising about the generation of the data, also pose important questions about
DOI: 10.1161/CIRCRESAHA.114.304676 Downloaded from http://circres.ahajournals.org/ at University of Texas--Austin on June 28, 2014
2
myocardial biology and the prospects of developing effective regeneration/repair protocols: Is the adult myocardium a self-renewing tissue? If so, what is the physiological significance of the self-renewal? Are the c-kit+ cells the regenerative/repair agents? If not, which are these regenerative agents? Clarification of the reason(s) for the reported discrepancies is of high priority, not only for cardiac biology but also for the future of the field of myocardial repair and regeneration. Discrepant and even contradictory results on a given topic are nothing new in science, even when the experiments are properly planned and the results accurately reported. In most cases the discrepancies are often due to a misinterpretation of the power of the experimental system(s) used which often leads to the over- and/or mis-interpretation of the results. The latter appears to be main issue with the van Berlo et al. paper. Not all Cre/LoxP cell-tracking systems provide reliable answers One perennial conundrum in cell biology has been the search for an efficient and accurate method of tracking the cell lineage origin as well as the fate of a given cell type, both during development and in adulthood. The development of the Cre/LoxP system of constitutive and conditional gene targeting together with trans- or knocked-in genes seemed the ideal procedure for fate mapping of specific cells. Inducible or regulated Cre-expressing mouse lines are increasingly popular because they significantly improve temporal and spatial targeting of genes (4). The most widely used inducible Cre lines utilise a Cre fused to a mutant ligand-binding domain of the oestrogen receptor (Cre-ER, mER-Cre-mER or CreERT2) where Cre-ER, mER-Cre-mER or Cre-ERT2 activation depends on the selective oestrogen receptor modulator tamoxifen (4). Although the constitutive and conditional Cre/loxP systems are both extremely powerful, they has several drawbacks that need to be properly controlled to avoid erroneous interpretation of observed results (4). As the van Berlo et al., work is exclusively based on the efficiency and fidelity of the Cre knock-in in the c-kit locus to track c-kit+ cell lineage fate, one must consider the main shortcomings of this recombination system that are summarized in Table 1. Therefore, considering these shortcomings, the conclusions reached with the Cre/lox system are only as reliable as the specific controls carried out to insure that the system performance is appropriate to unambiguously answer the questions addressed by the experiment. Table 2 summarizes the three controls that are particularly important. Unless these conditions are fulfilled the results of a Cre/lox experiment are un-interpretable. This is even more so when the results are negative because there is low or absent identifiable marked progeny of the target cells, as is the case of the results of van Berlo et al. These caveats are particularly important in light of the very complex and yet not fully elucidated c-kit gene regulation (6). In fact, all the c-kit transgenic or knock-ins produced to track c-kit expressing cells are far from closely recapitulating c-kit expression and function either in development or adult life (6,7). Does the c-kit/Cre knock-in recombine the reporter gene in the c-kit+ eCSCs? It is evident that both the constitutive and regulated Cre c-kit gene mutated mice lines used by van Berlo et al., are c-kit hypomorphs, most likely hemizygous null, which are equivalent to the W/+ mutant mice (8). While the typical signs of a c-kit hemizygous null allele (white spots on the belly, paws and tip of the tail) are not clearly reported in the paper, this conclusion is confirmed by the fact that the homozygous animals are not viable at birth and mostly die in utero. Whether the level of Cre expression
DOI: 10.1161/CIRCRESAHA.114.304676 Downloaded from http://circres.ahajournals.org/ at University of Texas--Austin on June 28, 2014
3
from the mutated allele is sufficient to produce recombination in all or most of the c-kit expressing cells is unknown, except that there is recombination in cells/tissues with high endogenous c-kit expression. The authors provided data showing recombination in several cell types, including myocardial cells, but not in the cells relevant to the results, i.e. the c-kit+ eCSCs. The reported recombination in total c-kit+ bone marrow cells and in myocyte-depleted c-kit+ cardiac cells is not relevant to the efficacy of the Cre/lox set-up because it is not specified which are the c-kit+ cell types recombined and which are not. In the constitutive Cre-IRES-nGFPxR26R-eGFP mice if the recombination were spatially and temporally correct, the majority (if not all) of blood cells in the adult should be labelled, as c-kit is expressed in all the embryonic cells with hematopoietic activity. Yet this piece of evidence is not provided. Furthermore, bone marrow and myocardial c-kit+ cells are complex cell mixtures with
