Undeniable Evidence That the Adult Mammalian Heart Lacks an Endogenous Regenerative Stem Cell
2018; Lippincott Williams & Wilkins; Volume: 138; Issue: 8 Linguagem: Inglês
10.1161/circulationaha.118.035186
ISSN1524-4539
AutoresBryan D. Maliken, Jeffery D. Molkentin,
Tópico(s)Mesenchymal stem cell research
ResumoHomeCirculationVol. 138, No. 8Undeniable Evidence That the Adult Mammalian Heart Lacks an Endogenous Regenerative Stem Cell Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBUndeniable Evidence That the Adult Mammalian Heart Lacks an Endogenous Regenerative Stem Cell Bryan D. Maliken, BA and Jeffery D. Molkentin, PhD Bryan D. MalikenBryan D. Maliken University of Cincinnati and Cincinnati Children's Hospital Medical Center, OH (B.D.M., J.D.M.) and Jeffery D. MolkentinJeffery D. Molkentin Jeffery D. Molkentin, PhD, Howard Hughes Medical Institute, Cincinnati Children's Hospital Medical Center, 240 Albert Sabin Way, Cincinnati, OH 45229. E-mail E-mail Address: [email protected] University of Cincinnati and Cincinnati Children's Hospital Medical Center, OH (B.D.M., J.D.M.) Howard Hughes Medical Institute, Cincinnati Children's Hospital Research Foundation, OH (J.D.M.). Originally published20 Aug 2018https://doi.org/10.1161/CIRCULATIONAHA.118.035186Circulation. 2018;138:806–808Article, see p 793Over the past 2 decades, reports of adult stem cells serving as mediators of cardiac regeneration of infarcted tissue have been a source of great hope in cardiovascular medicine. One of the most highly touted adult stem cell types was defined by expression of the cell surface receptor c-Kit, which in bone marrow marks the known hematopoietic progenitors that give rise to all immune and blood cells. With respect to the heart, publications in the early 2000s showed that injection of c-Kit+ cells, either bone marrow derived or derived from within the heart itself, was able to regenerate up to ≈70% of the infarcted rodent myocardium when injected into the injured area.1,2 Although independent laboratories were unable to replicate these early findings,3–5 the field nonetheless moved quickly to the clinic with an array of human trials, starting with bone marrow mononuclear cells and followed thereafter with expanded autologous cardiac stem cells or mesenchymal stem cells from various sources. Collective evidence from these trials has demonstrated that "adult stem cell" injection is largely safe in patients but that efficacy remains unclear, with some trials failing to meet primary end points.5 None of these injected cell types have demonstrated an ability to engraft and directly generate new myocardium in clinical applications, and today, the preponderance of recent evidence in rodent models fails to demonstrate physiologically relevant new cardiomyocyte generation with injection of any sort of presumed adult stem cell.6,7The assertion that the heart contains its own dedicated cardiomyocyte-producing stem cell is absolutely central to the entire cardiac regeneration–stem cell debate. If the heart truly lacks such an endogenous adult stem cell, this calls into question previous studies in which cardiac regeneration was reported such as from c-Kit+ cells isolated from the heart, selected, expanded, and reinjected back into the circulation, where they then homed to the heart and created new myocardium.6 Indeed, the US Food and Drug Administration recently summarized the state of affairs surrounding stem cell therapy for indications such as heart failure as follows:Outside the setting of hematopoietic reconstitution and a few other well-established indications, the assertion that stem cells are intrinsically able to sense the environment into which they are introduced and address whatever functions require replacement or repair—whether injured knee cartilage or a neurological deficit—is not based on scientific evidence.8In our opinion, the more recent and rigorous genetic approaches in mouse models9–12 such as the article by Li and colleagues13 in this issue of Circulation strongly demonstrate that the adult heart lacks a predetermined cardiomyocyte-producing stem cell. For example, to directly assess whether c-Kit+ cells are endogenous cardiac stem cells, multiple independent laboratories generated Kit locus genetically targeted mice in which both constitutive and inducible Cre recombinase–dependent lineage tracings were performed in the adult heart at baseline and with injury.9–11 The results of these studies showed that cardiomyocyte generation from a c-Kit+ cell lineage was an extremely rare event (<0.005%) that was several orders of magnitude below the known physiological turnover rate for cardiomyocytes.14 However, advocates of the c-Kit stem cell hypothesis raised concerns about the technical caveats of lineage tracing, specifically whether the Kit allele heterozygosity used in these approaches might hinder myogenic potential or whether lineage tracing mouse models are unable to mark very low c-Kit+–expressing cells that somehow have more "stemness."15 Li and colleagues provide definitive evidence with genetic lineage tracing that is not dependent on the Kit allele or any other allele that presumably marks a stem cell population. Instead, they used a dual Cre-Dre lineage tracing genetic approach in mice that conclusively shows that the adult heart does not generate new cardiomyocytes from nonmyocyte sources, whether c-Kit expressing or otherwise.13Summary of the ResultsMore specifically, Zhou's laboratory at the Shanghai Institute of Biochemistry and Cell Biology previously showed that traditional Cre-recombinase–driven lineage tracing systems can lead to misidentification of Kit-derived cardiomyocytes as a result of ectopic expression in preexisting cardiomyocytes.11 A similar concern arises with testing any putative cardiac stem cell candidate that relies on a specific marker gene for locus-dependent lineage tracing (Sca1, Abcg2, Isl1, BMi1, etc). To address this limitation, Zhou's group used an elegant dual genetic recombinase system showing that Kit-derived cardiomyocytes are never observed when the possibility of ectopic Kit allele expression is eliminated.12 In the group's most recent work,13 this finding is taken a step further, using the same dual recombinase lineage tracing system with a series of "interleaved genetic reporters." Using this more refined system, the authors first label all cardiomyocytes permanently, followed by a second wave of recombination induced in all other cell types, which showed that in no case were cardiomyocytes derived from noncardiomyocytes in the adult mouse heart at baseline or with injury.13The results are exceptionally rigorous not only because of the sophisticated genetic experimental design but also because of the high redundancy of experiments and convincing positive controls in both developmental and adult settings. To achieve multiple independent approaches to prelabel all cardiomyocytes, the authors used 2 different cardiomyocyte promoters (Tnnt2-Dre or Tnnt2-Cre and Tnni3-Dre) in various combinations with their ubiquitous nonmyocyte drivers that were based on targeting of the ubiquitous Rosa26 locus or the β-actin promoter (R26-iCre, R26-DreER, and Actb-Cre), which showed remarkable reproducibility with either interleaved reporter. More important, the differentiation of noncardiomyocytes into cardiomyocytes was readily observed during early heart development and in adult skeletal muscle where nonmyocyte satellite cells are known to contribute to new myofiber formation. Even with <100% efficiency of Rosa26 reporter allele recombination driven by either the Cre or Dre lines, there is no biological reason why the minor unlabeled population would selectively include all potential cardiomyogenic stem cells because the entire system is essentially "blind" to the labeling of nonmyocytes. Lastly, 9 different inducible Cre lines were used, several of which were generated for this report, again demonstrating that cardiac cell types, including endocardial cells, mesenchymal stromal cells, resident and activated fibroblasts, endothelial cells, pericytes, smooth muscle cells, and epicardial cells, are incapable of producing new cardiomyocytes in the adult heart.13Ramifications and ConclusionsThe primary biological ramification of the genetic evidence provided by Li and colleagues13 is that the adult heart lacks an endogenous cardiomyocyte producing stem cell. This conclusion may not be a surprise to many cardiac biologists and cardiologists because it is fairly well known that the heart is one of the least regenerative tissues in the adult mammal, with estimates of ≈1% cardiomyocyte nuclear DNA synthesis events per year, with true cellular division likely occurring at an even smaller frequency.14 However, the even greater ramification is that we should now reinterpret a portion of the previous cardiac literature in this area such as those reports claiming that cardiac-derived c-Kit+ cells broadly regenerate the heart with new cardiomyocytes.2,15,16 Moreover, it is highly doubtful that another putative adult stem cell type such as mesenchymal stromal cells, adipocyte progenitors, and fractionated bone marrow cells would be inherently more cardiogenic or reparative to the heart.The final and perhaps more controversial ramification of the work by Li and colleagues13 is that the field needs to potentially re-evaluate the current cadre of ongoing and planned clinical trials with cardiac cell transplantation because it is clear that we no longer understand the true mechanistic basis for this approach. It is also difficult to optimize such clinical trials for success if we lack a comprehensive biological understanding of how cell therapy works, which is especially germane in light of recent negative results from ongoing trials.5 For example, the results reported by Li and colleagues suggest that harvesting autologous c-Kit+ cells from the human heart or harvesting any other presumed progenitor cell type from the heart for clinical trials is of no greater mechanistic benefit in promoting cardiac rejuvenation than using cells derived from noncardiac sources. Indeed, animal studies and some clinical trials have shown a reparative benefit from cellular transplantation approaches from an array of seemingly unrelated cell types from many different sources.6–8 Thus, the potential "rejuvenating" benefit underlying cell therapy needs to be more rigorously investigated so that clinical trials can be better designed and optimized for efficacy in moving forward. Recent emerging hypotheses are that the injected/infused progenitor cells serve as a temporary source of paracrine factors that can have a reconditioning effect on the newly injured heart or that the injected/infused cells induce an acute inflammatory response as these cells die and are cleared, resulting in a secondary acute healing response. However, the results from Li and colleagues, in conjunction with other recent genetic approaches such as Kit allele lineage tracing,9–13 unequivocally demonstrate that cell therapy is not creating new cardiomyocytes, suggesting that we need to expand scientific efforts in the laboratory to continue elucidating the underlying mechanistic basis for cardiac cell therapy.Sources of FundingThis work was supported by grants from the National Institutes of Health (Dr Molkentin) and by the Howard Hughes Medical Institute to Dr Molkentin. B.D. Maliken was supported by National Institutes of Health National Heart, Lung, and Blood Institutes F30 grant HL137239-01, American Heart Association predoctoral grant 17PRE33410368, and National Institutes of Health T32 HL007752-23S1.DisclosuresDr Molkentin received significant research support from the National Institutes of Health (R37HL60562); however, no direct conflicts of interest related to this editorial are declared. B.D. Maliken reports no conflicts.Footnoteshttps://www.ahajournals.org/journal/circThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Jeffery D. Molkentin, PhD, Howard Hughes Medical Institute, Cincinnati Children's Hospital Medical Center, 240 Albert Sabin Way, Cincinnati, OH 45229. E-mail jeff.[email protected]orgReferences1. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. 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August 21, 2018Vol 138, Issue 8 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.118.035186PMID: 30359129 Originally publishedAugust 20, 2018 KeywordsEditorialsstem cellsPDF download Advertisement
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