doi: 10.1161/CIRCULATIONAHA.105.610287
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(Circulation. 2006;113:1275-1277.)
© 2006 American Heart Association, Inc.
Editorial |
You Can’t Judge a Book by Its Cover
From Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery, INSERM U 633, University Paris-Descartes, Faculty of Medicine, Paris, France.
Correspondence to Dr Philippe Menasché, Department of Cardiovascular Surgery, Hôpital Européen Georges Pompidou, 20 rue Leblanc, 75015 Paris, France.
Key Words: Editorials • heart failure • cells • myocardial infarction • stem cells
The current issue of Circulation reports 2 apparently conflicting studies1,2 pertaining to the highly controversial issue of transdifferentiation of bone marrow–derived cells into cardiomyocytes. The study by Iwasaki et al1 entailed intramyocardial injections of human CD34+ progenitors 20 minutes after the creation of a myocardial infarction in sex-mismatched, immunodeficient athymic mice. The results, assessed 28 days after transplantation, demonstrate a dose-dependent improvement in regional and global left ventricular function, a limitation of remodeling, and an increase in capillary density. These functional benefits are associated with a cardiac differentiation of the engrafted CD34+ cells demonstrated by a double immunofluorescent staining for human- and cardiac-specific markers. Expression of cardiac genes unraveled by real-time polymerase chain reaction is also taken as additional evidence for the cardiomyocytic conversion of the CD34+ progenitor cells, although this conclusion is compounded by the concomitant observation of fusion events between human and mouse cells demonstrated by fluorescent hybridization in situ, using probes specific for these 2 species. Differentiation of the CD34+ progenitors into smooth muscle and endothelial cells is also reported on the basis of similar double-staining immunofluorescent patterns. In contrast, the second article, by Gruh et al,2 does not demonstrate any conversion of human endothelial progenitor cells cocultured with neonatal rat cardiomyocytes. This study has carefully deciphered the potential causes of artifacts that may arise from the use of flow cytometry and conventional 2-dimensional immunofluorescence microscopy and lead to misinterpretations of phenotypic changes incurred by transplanted cells. Indeed, even 3-dimensional confocal microscopy failed to provide a conclusive interpretation for approximately 12% of the double-stained cells (ie, those expressing markers of both endothelial and cardiac lineages), thereby highlighting the caution with which these putative transdifferentiation events should be analyzed. Failure of additional real-time polymerase chain reaction to detect cardiac transcription factors further supports the authors’ skepticism about the ability of CD34+ progenitor cells to cross their lineage boundaries, at least in this setting of coculture experiments.
Articles pp 1311 and 1326
At first glance, the seemingly opposite conclusions of these 2 articles add to the confusion among clinicians about the capacity of bone marrow–derived cells to improve function of the infarcted myocardium through their conversion into cardiomyocytes. However, it is probably not meaningful to try to reconcile these data, as the 2 studies are not strictly comparable. First, despite some phenotypic overlap, the cell populations under investigation are different: Endothelial progenitor cells only comprise a minority of CD34-positive cells,3 whereas, correlatively, the CD34+ population studied by Iwasaki et al1 only contained 0.41% and 0.18% of cells staining positively for KDR and VE-cadherin, respectively, which are 2 hallmarks of endothelial progenitors. Second, the fate of cells cocultured in an in vitro setting probably does not predict accurately what occurs in vivo, where local cues, particularly in an infarcted area, may drive the differentiation of engrafted cells along cardiomyogenic pathways.4,5 Furthermore, to put these studies in a clinical perspective, it is relevant to note that only a very small amount of the 2 cell populations under investigation (
0.02% for true endothelial progenitors and 1% to 2% for CD34+ cells) is present in the mononuclear cell yield that is injected into the coronary arteries of the patients enrolled in the bone marrow cell therapy trials that have been implemented so far.
Thus, instead of opposing these 2 studies in a face-to-face fashion, it looks more constructive to try to extract from their content some key lessons that may be useful to better understand the mechanisms of action of cell therapy and harness its potential benefits for the management of infarcted myocardium and heart failure. At least 3 major messages are conveyed by these articles.
The first is a new word of caution about reliance on immunofluorescence. Indeed, the need for an arbitrary cutoff for positive staining, autofluorescence of infarcted tissue and cellular debris, and the resulting overlap of different fluorescent signals from adjacent cells can lead to mistaken interpretation of the fluorescence of engrafted cells as evidence for transdifferentiation. Although some have argued that it should not be a concern when adequate protocols of tissue labeling and detection are used,6 there is increased confidence that genetic markers provide a much more reliable way of tracking the fate of engrafted cells and of determining whether they have simply adopted the phenotype of cardiomyocytes as the result of their fusion with native muscle cells or have truly reprogrammed their genes to assume the new cell type. Of note, studies that have used this more robust genetically based tracking method have failed to document any conversion of bone marrow–derived cells into cardiomyocytes.7,8
Second, the fact that a given cell has taken on the shape of a cardiomyocyte and positively stains for cardiac-specific surface markers does not automatically mean that this cell has become a true cardiomyocyte.9 Such an assumption requires a more accurate characterization at the molecular and electrophysiological levels and the demonstration that the engrafted cells have connected with the host cardiomyocytes to form a functional syncytium, thereby allowing the graft to beat in synchrony with the remainder of the heart and, consequently, to efficiently contribute to its pump function. This view is supported by the observation that cocultured c-kit+ stem cells in neonatal cardiac myocytes express cardiac markers as well as Na+ and Ca2+ voltage-gated ion channels; however, these channels are not functional (on the basis of patch-clamp recordings) and consequently do not allow excitation-contraction coupling.10 Furthermore, even if one assumes that some CD34+ progenitors have the capacity of jumping the lineage barrier, the conversion phenomena appear to be extremely rare. In an earlier study, Jackson et al11 intravenously injected ß-galactosidase–expressing, bone marrow–derived hematopoietic stem cells into lethally irradiated wild-type mice and subsequently found only 0.02% of the recipient cardiomyocytes to be ß-galactosidase–positive in the infarcted hearts, whereas the prevalence of donor-derived endothelial cells, although higher, did not exceed 3.3%. A subsequent study by Nygren et al12 entailed reconstitution of lethally irradiated Rosa26 mice (which express ß-galactosidase ubiquitously) with bone marrow cells from transgenic green fluorescent protein (GFP)-expressing donors. Cell fusion (evidenced by a double positive staining for the 2 reporter genes) was demonstrated at a rate of 0.0065%, whereas transdifferentiation events (which would have been reflected by ß-galactosidase–negative, GFP-positive cells) could not be identified in a single animal. From this standpoint, it is noteworthy that although Iwasaki et al1 reported that both fusion and transdifferentiation accounted for the dose-dependent differentiation of CD34+ progenitors into multiple lineages, they did not specify the quantitative importance of these 2 events. This quantification was actually performed in a previous, closely related study in which human CD34+ cells were injected into infarcted mouse hearts. Flow cytometry analysis with the use of antibodies specific for human lymphocyte antigen and cardiac markers (either troponin T or NKx2.5) revealed that approximately 1% of the cells were double-stained, of which 70% contained in their nuclei both human male and mouse female chromosomes, suggesting cell fusion.13 This low percentage of cardiomyocytes of donor origin was obtained after transplantation of two million CD34+ cells, a dose 4-fold higher than the highest one used in the experiments conducted by Iwasaki et al.1 This issue of the phenotypic fate of engrafted cells is further complicated by the fact that it is strongly influenced by local cues which, in turn, probably depend on the nature of the host tissue (ie, normal versus infarcted myocardium), the timing of cell delivery, as well as on the species. In this setting, it is clinically relevant to note that in a nonhuman primate model of myocardial infarction entailing transplantation of autologous GFP-transfected CD34+ progenitors, no GFP-positive cardiomyocyte could be detected in the grafted tissue.14
The third message conveyed by these reports is that although the cardiomyogenic differentiation of CD34+ progenitors remains questionable and, at most, quantitatively very limited, the fact is that the intramyocardial implantation of these cells successfully improved postinfarction left ventricular function, a finding consistent with the observations made in the above-mentioned nonhuman primate experiments.14 Likewise, a recent study has shown that intravenous administration of bone marrow–derived male hematopoietic stem cells into female recipient mice improved left ventricular dimensions and function, as assessed by echocardiography and magnetic resonance imaging, despite the fact that less than 0.01% of the initially injected population could be identified 4 weeks later.15 Altogether, these data strengthen the concept that the functional benefits of bone marrow cell transplantation may be primarily mediated by paracrine mechanisms. One of the major targets of this paracrine signaling is likely to be angiogenesis (proliferation of preexisting vessels) and vasculogenesis (new blood vessel formation). Although the close temporal and physical proximity of their developmental pathways accounts for the fact that CD34+ progenitors can convert into endothelial cells (more commonly than into cardiomyocytes),13 there is compelling evidence that their angiogenic potential is primarily related to the secretion of growth factors triggering the development of new vessels.3,14 Cell-derived paracrine effects might also extend beyond angiogenesis and encompass limitation of native cardiomyocyte apoptosis,16 changes in extracellular matrix composition,17 or recruitment of endogenous cardiac stem cells. The latter hypothesis is indirectly supported by the observation that transplantation of human mesenchymal stem cells into the brain of mice increases expression of trophic factors, which stimulate the proliferation of endogenous neural stem cells.18 Assuming, however, that increased angiogenesis underlies most of the beneficial effects of CD34+ progenitors, this benefit must be weighted against the potential risk associated with their intracoronary infusion. This concern has been raised because of the link between angiogenesis and atherogenesis and is supported by the finding that CD34+ progenitors constitute the bulk of cells present in atherectomy specimens retrieved in-stent restenoses.19 This double-edged sword is indeed reflected by the paradoxic, not to mention confusing, observation that among current clinical trials, some investigate the effects of CD34+ intracoronary applications, whereas others test the effects of stents coated with anti-CD34+ antibodies.20 We hope that the outcomes of these trials will allow assessment of the risk/benefit ratio associated with the therapeutic application of this cell population, particularly when its upscale is obtained at the cost of cytokine mobilization.
More generally, these observations raise the basic question of whether improvement of left ventricular function is the primary objective assigned to cell therapy irrespective of the underlying mechanism or whether we should strive to meet this objective, at least in part through restoration of a new pool of functional cardiomyocytes. If one believes the latter paradigm, then alternate cell types such as extracardiac cardiogenic progenitor cells (for example, derived from fat tissue), the putative cardiac stem cells, or cardiac-committed embryonic stem cells are probably better candidates than endothelial or CD34+ progenitors. Given the amount of resources allocated to this field, it is likely that ongoing studies will ultimately identify the most effective type(s) of cells. In the meantime, the stimulating articles by Iwasaki et al1 and Gruh et al2 show that we still have to deal with this frustrating paradox of accumulating preclinical evidence for the efficacy of cell transplantation without really understanding the underlying mechanism of its action.
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Acknowledgments |
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Disclosure
I do not have a direct conflict of interest in relation with this editorial or the articles to which it refers, but my lab has received research support from Genzyme, a company for which I also act as a consultant.
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Footnotes |
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The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.
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References |
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2. Gruh I, Beilner J, Blomer U, Schmiedl A, Schmidt-Richter I, Kruse M-L, Haverich A, Martin U. No evidence of transdifferentiation of human endothelial progenitor cells into cardiomyocytes after coculture with neonatal rat cardiomyocytes. Circulation. 2006; 113: 1326–1334.
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