Challenging the Cardiac Differentiation of Bone Marrow Cells: A Clinical Perspective
Authors: Philippe Menasché
Journal: Molecular Therapy (2008)

The plasticity of adult bone marrow cells remains a controversial topic. The initial enthusiastic claims that these cells could "regenerate" the infarcted myocardium by converting into new cardiomyocytes1Orlic D Kajstura J Chimenti S Jakoniuk I Anderson SM Li B et al.Bone marrow cells regenerate infarcted myocardium.Nature. 2001; 410: 701-705Crossref PubMed Scopus (4714) Google Scholar have subsequently been dampened by reports that such an outcome was unlikely.2Murry CE Soonpaa MH Reinecke H Nakajima H Nakajima HO Rubart M et al.Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.Nature. 2004; 428: 664-666Crossref PubMed Scopus (1922) Google Scholar,3Balsam LB Wagers AJ Christensen JL Kofidis T Weissman IL Robbins RC Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium.Nature. 2004; 428: 668-673Crossref PubMed Scopus (1554) Google Scholar,4Nygren JM Jovinge S Breitbach M Säwén P Röll W Hescheler J et al.Bone marrow–derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.Nat Med. 2004; 10: 494-501Crossref PubMed Scopus (917) Google Scholar Indeed, the purported transdifferentiation of bone marrow cells has most often been based on immunohistochemical data, which are known to be potentially misleading because of the autofluorescence of infarcted tissue. More reliable cell-tracking methods, based on genetic tags, have led to the opposite conclusion—that bone marrow cells are in fact lineage-restricted.2Murry CE Soonpaa MH Reinecke H Nakajima H Nakajima HO Rubart M et al.Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.Nature. 2004; 428: 664-666Crossref PubMed Scopus (1922) Google Scholar,3Balsam LB Wagers AJ Christensen JL Kofidis T Weissman IL Robbins RC Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium.Nature. 2004; 428: 668-673Crossref PubMed Scopus (1554) Google Scholar,4Nygren JM Jovinge S Breitbach M Säwén P Röll W Hescheler J et al.Bone marrow–derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.Nat Med. 2004; 10: 494-501Crossref PubMed Scopus (917) Google Scholar Scherschel et al.,5Scherschel JA Soonpa MH Srour EF Field LJ Rubart M Adult bone marrow–derived cells do not acquire functional attributes of cardiomyocytes upon transplantation into peri-infarct myocardium.Mol Ther. 2008; 16: 1129-1137Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar using two-photon laser scanning microscopy (TPLSM), provide compelling evidence in this issue of Molecular Therapy that bone marrow cells actually lack a fundamentally important attribute of cardiomyocytes: the ability to develop cytosolic calcium transients in response to membrane depolarization. In this elegant and thoughtful study, the authors acutely transplanted three populations of green fluorescent protein–labeled bone marrow cells (low-density mononuclear cells and c-kit -enriched and highly enriched lineage-negative c-kit -positive cells) into a mouse model of myocardial infarction. After 9 to 10 days, the hearts were harvested, Langendorff-perfused, loaded with a calcium-sensitive fluorescent dye (rhod-2), and assessed by TPLSM imaging. Whereas host cardiomyocytes exhibited the expected cyclic calcium responses evoked by action potentials, none of the engrafted donor cells, regardless of phenotype, was found to be electrically excitable, either spontaneously or following field stimulation. Furthermore, the expression by the engrafted cells of the CD45 surface marker, along with the lack of positive staining for α-actinin, support a hematopoietic differentiation of the transplanted cells and their failure to convert into cardiomyocytes. Overall, these data complement those of Lagostena et al.,6Lagostena L Avitabile D De Falco E Orlandi A Grassi F Iachininoto M et al.Electrophysiological properties of mouse bone marrow c-kit cells co-cultured onto neonatal cardiac myocytes.Cardiovasc Res. 2005; 66: 482-492Crossref PubMed Scopus (40) Google Scholar who had already shown that bone marrow–derived c-kit -positive cells co-cultured with rat neonatal cardiomyocytes were not connected to surrounding cardiac myocytes through gap junctions and that, although they expressed Na+ and Ca2+ voltage-gated ion channels, these channels were functionally ineffective in that they failed to support excitation–contraction coupling. As with immunohistochemistry, TPLSM is fraught with potential artifacts, particularly when the donor and recipient cells are so close that the anatomic origin of the rhod-2 signals becomes of questionable reliability. Scherschel et al.5Scherschel JA Soonpa MH Srour EF Field LJ Rubart M Adult bone marrow–derived cells do not acquire functional attributes of cardiomyocytes upon transplantation into peri-infarct myocardium.Mol Ther. 2008; 16: 1129-1137Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar addressed this issue by avoiding screening calcium transients from donor cells that were closely juxtaposed to host cardiomyocytes, thereby eliminating the risk of misinterpreting as a calcium response what could actually have been due to fluorescence contamination. The results look convincing enough to support their conclusion that transplanted bone marrow cells do not acquire a cardiomyogenic phenotype during the short period that they remain identifiable in the grafted tissue. The next question for the clinician is to figure out whether it really matters. The answer is not unequivocal; it probably depends on the primary clinical indication of the cell-transplant procedure and, consequently, on the objective assigned to the cells. The easiest scenario is that of patients with disabling angina that becomes refractory to drugs and cannot be relieved by either a percutaneous or surgical revascularization procedure. The clear objective here is to increase angiogenesis, and there is robust and consistent evidence that bone marrow cells can successfully meet this objective because they behave as biofactories that release a wide array of angiogenic cytokines.7Kinnaird T Stabile E Burnett MS Lee CW Barr S Fuchs S et al.Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms.Circ Res. 2004; 94: 678-685Crossref PubMed Scopus (1267) Google Scholar The optimal type of bone marrow cells to be used in this indication remains an open question, but it is sound to select cells that feature the greatest angiogenic potential. From this standpoint, CD34+ progenitors look appealing. This assumption is supported by the encouraging results of a phase I–II trial8Losordo DW Schatz RA White CJ Udelson JE Veereshwarayya V Durgin M et al.Intramyocardial transplantation of autologous CD34+ stem cells for intractable angina: a phase I/IIa double-blind, randomized controlled trial.Circulation. 2007; 115: 3165-3172Crossref PubMed Scopus (487) Google Scholar in individuals with intractable angina, results that await confirmation by an ongoing larger randomized controlled study. A major caveat, however, is that patients with coronary artery disease often have functionally defective bone marrow cells, which may clearly compromise the ultimate clinical outcome.9Assmus B Fischer-Rasokat U Honold J Seeger FH Fichtlscherer S Tonn T et al.TOPCARE-CHD Registry. Transcoronary transplantation of functionally competent BMCs is associated with a decrease in natriuretic peptide serum levels and improved survival of patients with chronic postinfarction heart failure: results of the TOPCARE-CHD Registry.Circ Res. 2007; 100: 1234-1241Crossref PubMed Scopus (187) Google Scholar This issue could be addressed by the use of mesenchymal stem cells that also feature an angiogenic potential but in addition are credited with an immune privilege.10Ryan JM Barry FP Murphy JM Mahon BP Mesenchymal stem cells avoid allogeneic rejection.J Inflamm (Lond). 2005; 2: 8Crossref PubMed Scopus (702) Google Scholar This could offer the advantage of use as a well-characterized and functionally tested allogeneic, readily available "off-the-shelf" cell product. Regardless of the cell type to be used, one should not be very worried about the inability of the bone marrow cells to adopt a cardiomyogenic phenotype, as demonstrated by Scherschel et al.,5Scherschel JA Soonpa MH Srour EF Field LJ Rubart M Adult bone marrow–derived cells do not acquire functional attributes of cardiomyocytes upon transplantation into peri-infarct myocardium.Mol Ther. 2008; 16: 1129-1137Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar because this is not what is expected from them in this specific indication. The situation of individuals with acute myocardial infarction is more complex. In Western countries, most such patients undergo early reperfusion of the infarct-related coronary artery, and the major objective is not to increase the immediate postprocedural ejection fraction (the primary end point of all cardiac cell–therapy clinical trials conducted so far in this indication) but rather to prevent late left ventricular remodeling, which is associated with a poor outcome.11St John Sutton M Pfeffer MA Plappert T Rouleau JL Moyé LA Dagenais GR et al.Quantitative two dimensional echocardiographic measurements are major predictors of adverse cardiovascular events after acute myocardial infarction: the protective effects of captopril.Circulation. 1994; 89: 68-75Crossref PubMed Google Scholar Cells that can be used for this objective should meet three major requirements: immediate availability, to avoid any delay in therapy; a size small enough to allow safe intracoronary infusions; and the ability to interfere with the mechanisms of remodeling. Theoretically, bone marrow cells match these criteria. More specifically, they release proangiogenic and antiapoptotic proteins that have been experimentally shown to reduce left ventricular dilatation.12Uemura R Xu M Ahmad N Ashraf M Bone marrow stem cells prevent left ventricular remodeling of ischemic heart through paracrine signaling.Circ Res. 2006; 98: 1414-1421Crossref PubMed Scopus (553) Google Scholar These expectations, however, sharply contrast with the results of the first wave of clinical trials that have, overall, demonstrated a statistically significant (but clinically irrelevant) 3% increase in ejection fraction but have failed to show a reduction in end-diastolic volumes in patients receiving intracoronary infusions of bone marrow–derived mononuclear cells compared with the controls.13Lipinski MJ Biondi-Zoccai GG Abbate A Khianey R Sheiban I Bartunek J et al.Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction.J Am Coll Cardiol. 2007; 50: 1761-1767Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar This failure could be due to several causes, including individual variability in cell functionality, inadequate timing of delivery, and insufficient extent of engraftment, and it can be reasonably predicted that if each of these issues could be successfully addressed, the benefits of the procedure would probably be optimized. Again, however, these expectations are not called into question by the data of Scherschel et al.,5Scherschel JA Soonpa MH Srour EF Field LJ Rubart M Adult bone marrow–derived cells do not acquire functional attributes of cardiomyocytes upon transplantation into peri-infarct myocardium.Mol Ther. 2008; 16: 1129-1137Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar in that the objective assigned to the cells is not to substitute for the missing native cardiomyocytes but rather to paracrinally trigger endogenous signaling pathways leading to enhanced myocardial tissue salvage. Such is no longer the case for individuals with heart failure resulting from extensively scarred myocardial areas. Although the results have not been totally negative, the few randomized bone marrow14Hendrikx M Hensen K Clijsters C Jongen H Koninckx R Bijnens E et al.Recovery of regional but not global contractile function by the direct intramyocardial autologous bone marrow transplantation: results from a randomized controlled clinical trial.Circulation. 2006; 114: I101-I107PubMed Google Scholar,15Stamm C Kleine HD Choi YH Dunkelmann S Lauffs JA Lorenzen B et al.Intramyocardial delivery of CD133+ bone marrow cells and coronary artery bypass grafting for chronic ischemic heart disease: safety and efficacy studies.J Thorac Cardiovasc Surg. 2007; 133: 717-725Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar,16Assmus B Honold J Schachinger V Britten MB Fischer-Rasokat U Lehmann R et al.Transcoronary transplantation of progenitor cells after myocardial infarction.N Engl J Med. 2006; 355: 1222-1232Crossref PubMed Scopus (949) Google Scholar transplantation trials have had only marginally efficacious clinical outcomes. These data suggest that the paracrine mechanisms outlined above may still be operative through increased neovascularization, boosting of recipient cell-survival pathways, and changes in the composition of the extracellular matrix leading to improved scar elasticity,12Uemura R Xu M Ahmad N Ashraf M Bone marrow stem cells prevent left ventricular remodeling of ischemic heart through paracrine signaling.Circ Res. 2006; 98: 1414-1421Crossref PubMed Scopus (553) Google Scholar but also that these effects are not powerful enough to substantially impact patient outcomes. From this standpoint, the observations of Scherschel et al.5Scherschel JA Soonpa MH Srour EF Field LJ Rubart M Adult bone marrow–derived cells do not acquire functional attributes of cardiomyocytes upon transplantation into peri-infarct myocardium.Mol Ther. 2008; 16: 1129-1137Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar highlight the limits of bone marrow cells when the primary objective is the replacement of the dead cardiomyocytes with the transplanted cells. To be effective in this context, the latter must engraft into the recipient myocardium in large amounts—because of the extent of areas to be regenerated (the cardiomyocyte deficit has been estimated in the range of one billion cells)—and establish electromechanical connections with the neighboring cardiomyocytes to form a functional syncytium. This is a prerequisite for the cellular graft to beat in synchrony with the remainder of the heart and thus directly contribute to its pump function. The data from Scherschel et al.5Scherschel JA Soonpa MH Srour EF Field LJ Rubart M Adult bone marrow–derived cells do not acquire functional attributes of cardiomyocytes upon transplantation into peri-infarct myocardium.Mol Ther. 2008; 16: 1129-1137Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar clearly demonstrate that bone marrow cells are unable to meet this objective, and there is now mounting evidence that the cells that are most suitable for achieving this are those that recapitulate a cardiomyogenic differentiation program. The choice is then limited. The serious doubts about the possibility of therapeutically exploiting putative cardiac stem cells in adults with ischemic heart disease17Pouly J Bruneval P Mandet C Proksch S Peyrard S Amrein C et al.Cardiac stem cells in the real world.J Thorac Cardiovasc Surg. 2008; 135: 673-678Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar justify exploration of the potential benefits of cardiac progenitors derived from human embryonic stem cells,18Tomescot A Leschik J Bellamy V Dubois G Messas E Bruneval P et al.Differentiation in vivo of cardiac committed human embryonic stem cells in postmyocardial infarcted rats.Stem Cells. 2007; 25: 2200-2205Crossref PubMed Scopus (135) Google Scholar primarily because these cells feature the fundamental attribute that is lacking in bone marrow cells: the ability to form a drug-responsive electromechanically integrated functional syncytium in the recipient myocardium.19Kehat I Khimovich L Caspi O Gepstein A Shofti R Arbel G et al.Electromechanical integration of cardiomyocytes derived from human embryonic stem cells.Nat Biotechnol. 2004; 22: 1282-1289Crossref PubMed Scopus (744) Google Scholar,20Xue T Cho HC Akar FG Tsang SY Jones SP Marban E et al.Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers.Circulation. 2005; 111: 11-20Crossref PubMed Scopus (405) Google Scholar There is increasing evidence that a "one cell fits all" concept is not viable; the choice of the optimal cell type should be tailored to the primary clinical indication. The findings of Scherschel et al.5Scherschel JA Soonpa MH Srour EF Field LJ Rubart M Adult bone marrow–derived cells do not acquire functional attributes of cardiomyocytes upon transplantation into peri-infarct myocardium.Mol Ther. 2008; 16: 1129-1137Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar are thus extremely helpful for fine-tuning the customization of the cell type to patients' specific needs. More specifically, although they do not exclude the common view that bone marrow cells may be reasonable candidates when a purely paracrine effect is expected, they also clearly indicate that, because of the inability of these cells to display action potential–evoked cyclic rises in cytosolic calcium, the cells fail to acquire a true cardiomyogenic phenotype and, as such, are not expected to be effective in heart-failure patients who need repopulation of their infarcted myocardial areas by functionally integrated contractile cells.