Cardiac Stem Cells and Progenitors: Developmental Biology and Therapeutic Challenges

Abstract

Discovery of embryonic and adult stem cells holds great promise for the treatment of cardiovascular disease. Embryonic stem (ES) cells differentiate into cardiac myocytes as well as multiple other cell lineages in vitro and when transplanted into the heart. However, their clinical application is limited by their pleuripotent nature, capacity to form teratomas and ethical concerns. Several groups have reported that bone marrow-derived cells differentiate into cardiac myocytes, though these findings remain controversial requiring further examination. Nevertheless, injection of bone marrow-derived cells in the setting of experimental models of myocardial infarction has demonstrated functional benefit. Similarly, small phase I/II clinical trials of bone marrow derived mononuclear cell infusions in the setting of acute myocardial infarction have generally demonstrated short term functional benefit. Most recently, several distinct populations of resident stem cells have been identified in the heart. Further elucidation of cardiovascular stem cell biology is a crucial first step in translating the promise of cardiac stem cells and progenitors into the clinical reality.

Introduction

During the latter half of the 20th century the translation of basic science and clinical epidemiology led to elucidation of the root causes and pathophysiology of atherosclerotic heart disease (1). The explosion of basic knowledge coupled with advances in drug discovery and medical technologies ultimately led to dramatic reduction in age-adjusted death rates from cardiovascular disease. Despite these advances, cardiovascular diseases remain the leading cause of mortality in western societies and the prevalence of disease is projected to increase further as the population ages. At the dawn of the 21^st century, definition of the human genome promises to provide important insights into the molecular and genetic basis of cardiovascular diseases. Moreover, the discovery of previously unrecognized plasticity in both embryonic and adult cells and their capacity to differentiate and/or transdifferentiate into multiple distinct cell lineages offers promise for the treatment of “irreversible conditions” including myocardial infarction, cardiomyopathy and congestive heart failure.

A great deal has been written about the application of stem cell therapies for cardiovascular disease and the reader is referred to several excellent reviews on this subject (2–5). Because of the explosion of knowledge in this area of research, indeed over one hundred abstracts on cardiovascular stem cell therapeutics were presented at the annual scientific sessions of the American Heart Association, the information discussed will be outdated, when this review is ultimately published. For this reason, I have attempted to summarize the concepts underlying the biology of cardiovascular stem cells and to highlight the principles that should guide the application of stem cell therapies in the clinical setting.

Stem Cell Biology

Hematopoietic stem cells (HSCs) were first identified in the 1960’s, but were not fully purified or characterized until the late eighties (6,7). The careful characterization of HSCs ultimately led to successful bone marrow transplantation in patients with lymphomas and leukemias. Through this experience a rigorous definition of “stem cell” was established. The four properties stem cells must exhibit include the capacity: 1) to undergo multiple sequential self-renewing cell divisions, 2) for single stem cell-derived daughter cells to differentiate into more than one cell type, 3) to functionally repopulate the tissue of origin in a damaged recipient, 4) to contribute differentiated progeny in vivo in the absence of tissue damage. It is important that this definition be considered before concluding that a cell is in fact a “cardiac stem cell”.

By contrast, the definition of “cardiac progenitor” or “cardioblast” has not been rigorously defined. Skeletal myoblasts represent a population of cells specified to the skeletal muscle cell fate, but which have not terminally differentiated into cells expressing the full repertoire of skeletal muscle-specific contractile proteins. These cells are by definition not multipotent and may be defined functionally by a specific transcriptional program that requires expression of skeletal muscle cell-specific transcription factors, including MyoD (8). Therefore cardiac progenitors or cardioblasts, if they exist, would represent a population of cells specified to the cardiac myocyte cell lineage prior to their (terminal) differentiation into a fully differentiated cardiac myocytes. Cardiac myocytes, in turn, express the full repertoire of differentiated cardiac-specific or cardiac-restrictive markers, such as the cardiac-specific isoform of troponin I (cTnI).

Over the past decade, fundamental insights into the molecular and developmental programs regulating cardiac myocyte differentiation from pre-cardiac mesoderm have been gained through the identification and characterization of a set of cardiac myocyte-restricted transcription factors (for review see (9,10)). However, in contrast to the skeletal muscle cell lineage, cardiac-specific transcription factors have not been identified. This has led to confusion distinguishing cardiac progenitors from other mesodermally (and endodermally)-derived cell lineages. The three transcription factors most often cited as markers of the cardiac lineage are GATA4, Nkx2.5 and MEF2C. However, none of these markers is restricted solely to the cardiac myocyte cell lineage. Nevertheless, by analogy to the skeletal muscle cell lineage, a pre-specified cardiac progenitor, or cardioblast, would be anticipated to express alone or in combination GATA4, Nkx2.5 and/or MEF2C. At the same time, cardioblasts would not express definitive markers of other cell lineages. When evaluating the origin and developmental potential of putative cardiac progenitors, or stem cells, it is critically important to demonstrate the expression of cardiac markers as well as the (lack of) expression of other mesodermal, endodermal and ectodermal markers. Many reports have assigned cells to the cardiac muscle cell lineage and/or concluded that cells represent cardiac stem cells or cardiac precursors because they express one or more cardiac markers. However, in many cases, a full survey of non-cardiac markers was not performed, complicating interpretation of these data.

By contrast, differentiated cardiac myocytes have been anatomically, biologically and physiologically well characterized. These cells, which until recently were considered terminally differentiated, express a unique set of cardiac-restricted contractile proteins, enzymes and cell surface receptors. Cardiac myocytes also express cardiac-restricted transcription factors including GATA4, Nkx2.5 and MEF2C (for review see (11)). Molecular programs distinguish the spatial configuration and functional properties of cardiac myocytes. Specific contractile protein isoforms are expressed in the atria (i.e. MLC2A) and others in the ventricle (i.e. MLC2V). Transcription factors restricted to the developing right ventricle and the left ventricle have been identified (12,13). Anatomically, cardiac myocytes exhibit striations representing sarcomeres, which are distinct from the contractile apparatus of skeletal muscle. Cardiac myocytes electrically and mechanically couple to adjacent cardiac myocytes through characteristic gap junctions. Fetal and neonatal cardiac myocytes generally contain a single nucleus, while the majority of adult cardiac myocytes are bi-nucleated. Physiologically, cardiac myocytes exhibit spontaneous electrical depolarization with a characteristic action potential. The electrophysiological properties of cells in the cardiac conduction system differ fundamentally from other myocytes. With respect to the potential therapeutic application of cardiac stem cells, it is important to recognize that embryonic, neonatal and adult cardiac myocytes differ fundamentally with respect to their respective patterns of gene expression, sarcomeric structure and electrophysiological properties.

When considering the application of stem cells and/or cardiac progenitors for the treatment of cardiovascular disease, it will be critically important to define precisely the biology and developmental potential of the transplanted cells. Is the cell clonal or does it represent a population of diverse cells and/or distinct cell lineages? Is the cell multipotent or pleuripotent? If so, what are the risks of differentiation into non-cardiac myocytic cell lineages? Are the cells to be transplanted terminally differentiated? If not, what is their replicative capacity and does it need to be controlled? Does it matter if the transplanted cell has a fetal cardiac rather than postnatal cardiac phenotype? Will the transplanted cells functionally couple to myocytes in the host heart? Understanding the cell biology of the cardiac stem cell or progenitor as well as the receptive capacity of the host heart will be crucial to the safe and effective application of regenerative therapies for cardiovascular disease.

Embryonic Stem Cells

Since the mid-eighties, it has been known that mouse embryonic stem (ES) cells differentiate into cardiac myocytes during in vitro differentiation into cystic embryoid bodies (14). More recently, it has been reported that human ES cells possess the capacity to differentiate into cardiac myocytes in vitro and when injected into adult mouse hearts (15–25). ES cells can be expanded in vitro and retain their capacity to differentiate into cardiac myocytes (14). ES cell-derived cardiac myocytes most closely resemble embryonic cardiac myocytes and express the complete repertoire of cardiac-restricted transcription factors including GATA4, Nkx2.5, MEF2C, and Irx4 (26). ES cell-derived cardiac myocytes beat spontaneously in culture and express contractile proteins and ion channels (17,19). Physiologically these cells exhibit chronotropic responses and flux calcium (27–29). The ultrastructural of ES cell-derived cardiac myocytes most closely resemble fetal or neonatal cardiac myocytes expressing a subset of embryonic myofibrillar genes and forming poorly organized sarcomeres (26,30).

It has been proposed that human embryonic stem cells may be therapeutically efficacious in the treatment of various forms of congenital heart disease, though the application of ES cells to treat congenital heart disease will require further characterization of the molecular program underlying cardiac differentiation and morphogenesis. The majority of translational research has focused on the application of ES cells to treat myocardial infarction, heart failure and cardiomyopathy (for review see (31)). ES cell-derived cardiac myocytes fuse and form gap junctions with resident cardiac myocytes when injected into the intact adult heart (16,29,32). However, a major functional limitation of pleuripotent ES cells is their capacity to differentiate into multiple distinct cell lineages (2). This has led to efforts to restrict the developmental potential of ES cells to the cardiac myocyte cell lineage. Several approaches have been tried, including forced expression of one or more cardiac-restricted transcription factor (33), exposing ES cells to various combinations of growth factors (34) and co-culturing ES cells with neonatal or fetal cardiac myocytes (34). Scalable production of genetically-modified ES cells engineered with a transgene consisting of a cardiac-specific promoter driving the aminoglycoside phosphotransferase (Neo^R) gene has been reported (26,30,35–37). However, these genetically-targeted ES cells have gone through one, or more, rounds of selection increasing the risk of mutagenesis.

Despite these limitations and potential risks, several groups have reported functional benefits of embryonic stem cell infusions in the setting of experimental myocardial infarction in animals (34,38–40). Genetically-tagged mouse ES cells engrafted in the setting of a rat myocardial infarction with concomitant improvement in left ventricular ejection fraction (34,38–40). However, the efficiency of ES cell engraftment and/or differentiation into cardiac myocytes was not reported. As such, the functional benefits observed may, or may not, have resulted from repopulation of the infarcted tissue by ES cell-derived cardiac myocytes.

With respect to their clinical application, it is noteworthy that murine and human ES cells may be passaged and expanded ex vivo, and give rise to cardiac myocytes, closely resembling neonatal cardiac myocytes. Moreover, ES cell-derived myocytes have the contractile and electrophysiological properties of (neonatal) cardiac myocytes and functionally couple to cardiac myocytes when implanted into allogeneic hosts. However, beyond the ethical concerns that have limited the use of embryonic stem cells in the clinical setting, significant practical issues remain to be addressed. The capacity of ES cells to differentiate into multiple distinct cell lineages remains a barrier to their clinical application at present. Moreover, in animal models, ES cells transplanted into the myocardium may give rise to teratomas (2). Moreover, in vivo transfer of human ES cells into skeletal muscle gives rise to teratomas (41). Finally, ES cells are potentially immunogenic (42). Further science focusing on restricting the developmental potential of ES cells is clearly warranted.

Bone Marrow-Derived Cardiac Stem Cells

The clinical observation of a large percentage (18%) of host-derived cardiac myocytes in transplanted human hearts led to the hypothesis that cells of bone marrow origin may possess the capacity to migrate to the heart, engraft, and differentiate into the cardiac myocyte cell lineage (43). However, subsequent reports assessing the frequency of host-derived cardiac myocytes vary widely. Several groups have reported that the contribution of host-derived myocytes in transplanted hearts is an extremely rare event, occurring on the order of only one per 10³ or 10⁴ cardiac myocytes (44–46). By contrast, host-derived, vascular smooth muscle cells and endothelial cells were readily observed in these transplanted hearts. If in fact, this lower estimate proves to be correct, then host-derived myocytes may only represent rare cell fusion events between bone marrow-derived circulating cells and the allogeneic cardiac myocytes. There is no obvious explanation for these differences, though most likely they resulted from differences in the methods utilized to identify and quantify host cells in the transplanted heart.

Consistent with the hypothesis that bone marrow-derived cells differentiate into cardiac myocytes, Anversa and colleagues reported that a sub-population of bone marrow-derived cells possesses stem cell-like properties and differentiate into cardiac myocytes. These stem cells are lineage negative (Lin⁻), they do not express the cell surface markers CD34, CD45, CD20, CD45RO and CD8, but do express the hematopoietic stem cell marker, c-kit (c-kit^Pos). Infusion of these cells, partially reconstitutes the injured myocardium and improves ventricular function in animal models of ischemia and reperfusion (47–49). Importantly, GFP-tagged lin⁻c-kit^POS cells were demonstrated to differentiate into cardiac myocytes. Subsequently, Anversa and colleagues reported that injection of the stem cell mobilizing factors SCF and G-CSF prior to and following experimental infarction in the mouse leads to myocardial regeneration and improves survival (50). However, the conclusion that bone marrow-derived lin⁻c-kit^POS cells differentiate into cardiac myocytes has been challenged by several independent groups of investigators. Three distinct groups, utilizing methodologies in which bone-marrow derived cells were genetically-tagged failed to demonstrate the differentiation of lin⁻c-kit^POS cells into cardiac myocytes (51–53). Instead, they observed that in the heart, genetically-tagged bone marrow cells differentiate into hematopoietic and granulocytic cell lineages. However, most recently, Losordo and coworkers reported functional benefit of human bone marrow-derived Lin⁻c-kit^Pos stem cell infusion in a nude rat animal model of myocardial infarction (54). In addition, Hamano and colleagues, reported that injection of CD117+ bone marrow cells exposed to TGFβ results in functional benefit in mouse infarct model (55). It remains possible that these divergent results reflect differences in the experimental methodologies employed, though extreme care was taken to reproduce the experimental conditions employed in the original studies. For now, the case that bone marrow-derived stem cells possess the capacity to functionally repopulate the damaged heart with cardiac myocytes requires further validation.

Moreover, many critical issues directly relevant to the eventual successful application of bone marrow-derived stem cell in the clinical setting remain to be addressed, including: 1) the efficiency of stem cell engraftment in the heart, 2) optimization of methods to transfer cells to the heart (i.e. intra-myocardial vs intra-coronary vs intravenous), 3) differences in the capacity of distinct stem cell populations to engraft in the heart, 4) optimized timing of stem cell infusion in animal models (and patients) with acute myocardial infarction and other conditions, 5) the long term survival of cells that engraft in the heart, 6) the capacity of engrafted cells to differentiate into cardiac versus non-cardiac cell lineages, 7) the capacity of engrafted cells to electrically couple in the heart and their arrhythmogenic potential, and finally, 8) the fate of cells that do not engraft in the heart and potential systemic complications related to these cells. Further basic and translational science is clearly warranted characterizing the biology of bone marrow-derived cardiac stem cells, optimizing methods of cell delivery to the heart and assessing the benefits and risks of bone marrow-derived cell transfusions in the clinical setting.

Clinical Trials of Bone-Marrow Cells in Acute Myocardial Infarction

Remarkably, even as controversy raged regarding the capacity of bone marrow derived stem cells to differentiate into cardiac myocytes, phase I/II clinical trials examining the safety and efficacy of bone marrow-derived cell infusion in patients with acute myocardial infarction were begun (5,56–62). Table 1 summarizes the published phase I/II clinical trials of autologous bone marrow cell transfer in the setting of acute myocardial infarction. Of note, these human studies differed from the proof-of-concept studies in animals in several fundamental respects. First, the majority of these trials utilized relatively un-purified populations of bone marrow mononuclear cells (BMMNCs) obtained after patients presenting with acute myocardial infarction. Less than 0.1% of these cells represent stem cells. Of note, none of these trials utilized the Lin⁻c-kit^Pos cells described above. Second, in the majority of these trials, the BMMNCs were injected via a coronary artery (usually the infarct-related artery) as opposed to directly into the myocardium (which was done in the animal studies). This obviously could impact on the efficiency of cell engraftment. As such, the results of clinical trials may differ from the proof-of-concept studies in animals not because of failure of the animal models to translate, but rather because the clinical trials do not recapitulate the proof-of-concept studies performed in animals.

TABLE 1.

Phase I/II Clinical Trials of Autologous Bone Marrow Cells for Acute Myocardial Infarction

Investigators	Phase	Cells	Admin.	(N)	Controls	Endpts.	Conclusions
Li et al. (62)	I	BMCs	Myoc (CABG)	5	N	ET, N, Sx (1 year)	Safe, “improvement”
Fernandez-Aviles et al. (56)	I	BMCs	Intracoronary AMI/PCI	33	Y	Scin, Sx, Echo (6 months)	Imp. Reg. WM, EF
							No change LVEDV
Strauer et al. (58)	I/II	BMCs	Intracoronary	20	Y	Echo, Scin	Imp. Reg. WM
							No change EF
Stamm et al. (57)	I	AC133+ BMCs	Myoc (CABG)	12	N	Scin, Echo (12 days)	Safe, Imp. LVEDV, EF, Perf.
Assmus et al. (60)	I	BMCs CPCs	Intracoronary Post-AMI	59	N	LV Angio, MRI, survival (4 months)	No diff survival., Imp EF and MRI over time (no control)
Wollert et al. (59)	I/II	BMCs	Intracoronary AMI/PCI	60	Y	MRI (EF) (6 months)	Significantly improved EF at 6 mo with no benefit at 1 y

Abbreviations: BMCs-bone marrow cells; CPCs-circulating progenitor cells; Myoc-Intra-myocardial injection; AMI-acute myocardial infarction; CABG-coronary artery bypass graft; ET-exercise time; N-nuclear study; Sx-symptoms; Scin-Nuclear scintigraphy; Echo-echocardiography; MRI-magnetic resonance imaging; WM-wall motion; EF-ejection fraction; LVEDV-left ventricular end diastolic volume; Perf-nuclear perfusion imaging.

Moreover, there are significant limitations in the experimental design of each of these studies that must be considered carefully. First, the majority of trials did not include placebo controls and the investigators were not blinded. Sample size is also a problem. The largest study reported to date (BOOST) included only 60 patients (59). As with other phase I clinical trials, these studies were designed to test safety, and did not have the statistical power to detect efficacy. In two studies, patients received injections of bone marrow-derived cells at the time they were undergoing coronary artery bypass graft (CABG), making it impossible to distinguish beneficial effects attributable to the BMMNC infusions from the benefits of CABG (57,63). In addition, the endpoints utilized were relatively “soft”, generally measuring only short term changes in ventricular function and geometry rather than survival. Despite these caveats, each study revealed at least short term improvements in ventricular function and/or geometry following BMMNC injection in the setting of acute myocardial infarction.

It is important to recognize that several mechanisms other than differentiation of stem cells into cardiac myocytes could explain these functional benefits of BMMNC infusion in the setting of acute myocardial infarction. Infusion of bone marrow cells may include cells and/or give rise to inflammatory cells (lymphocytes or monocytes) that mediate critical functions in the reparative process in the peri-infarct setting. Moreover, differentiation of bone marrow-derived cells into fibroblasts that secrete collagen and extracellular matrix could alter the geometry of the scar following myocardial infarction and improve cardiac remodeling. Alternatively, there is strong evidence that the bone marrow contains a resident population of hemangioblasts and/or endothelial progenitor cells (EPCs) with angiogenic potential (for review see (64,65)). Improved tissue perfusion of the ischemic heart has been shown to improve indices of contractile performance. Finally, it is possible that the transplanted BM cells secrete a paracrine factor that activates a population of resident cardiac stem cells (see below). The totality of available experimental and clinical data strongly supports the hypothesis that these “alternative mechanisms”, account for some, if not most, of the functional benefit of stem cell infusions in the setting of acute myocardial ischemia and infarction.

Drug approval in the United States requires rigorous examination of the pharmacokinetics and toxicities associated with each investigational compound. This includes precise definition of dose-response and assessment of systemic toxicity. It is therefore remarkable that none of the phase I/II clinical trials of BMMNCs mandated precise characterization of the cells injected, measurement of the efficiency of cell uptake and engraftment by the heart and/or bio-distribution and elimination from the body.

Moreover, while methodologically challenging, the clinical trials performed to date have not provided any insights into the fate of cells injected in the heart or their capacity to differentiate into cardiac myocytes and/or other cell lineages. Collection of these quantitative and (if possible) mechanistic data is crucial to the rational design of future clinical trials and the eventual safe application of bone marrow-derived cells in the setting of myocardial infarction and ischemic heart disease.

These issues should be considered carefully in future clinical trials. It is critically important that clinical investigators in this field optimize the design of clinical trials to include: 1) precise characterization of injected cells, 2) requisite placebo controls, and 3) quantitative measures of cellular bio-distribution, engraftment and survival. At the same time, further translational studies are required to optimize methods of cell delivery and engraftment and to determine the fate of cells that engraft in the heart and other tissues.

Resident Populations of Cardiac Stem Cells and Progenitors

Over the past sixty years, the paradigm was established that shortly after birth cardiac myocytes become post-mitotic, losing their replicative capacity. Recently, several groups of investigators have challenged this paradigm and reported that the postnatal heart includes niches of cardiac stem cells and/or cardiac progenitors with the capacity to replicate and differentiate into fully differentiated cardiac myocytes (66–77). Piero Anversa and colleagues first described niches of lin⁻c-kit^POS cells in the adult rat (and human) heart (70,75). These lin⁻c-kit^POS cells are approximately 1/10^th the size of adult cardiac myocytes. When isolated by repeated panning or FACS sorting, 7–10% of these cells expressed the early cardiac-restricted markers GATA4, Nkx2.5 and MEF2 (75). As discussed above, expression of one or more of these markers does not definitively mark a cell as cardiac in origin, but does support this conclusion. These lin⁻c-kit^POS clones appear to be multipotent, differentiating into cells resembling cardiac myocytes, smooth muscle cells and endothelial cells. Of note, the population of cells expressing cardiac markers did not exhibit identifiable sarcomeres suggesting that at least ex vivo they represent very immature cardiac myocytes, or a mesodermal derivative, which may only be pushed in the direction of a cardiac myocyte. However, it is noteworthy that when the lin⁻c-kit^POS cells were injected into the hearts of syngeneic rats following experimental myocardial infarction, partial reconstitution of the myocardium was observed (75). As discussed above, whether the functional improvement in ventricular function resulted primarily from differentiation of the lin⁻c-kit^POS cells into cardiac myocytes or an alternative mechanism remains to be determined.

Schneider and Garry and coworkers identified a distinct resident population of stem cell antigen (Sca)-1-positive stem cells (66,76). These cells also express telomerase reverse transcriptase that has been associated with the capacity for self-renewal. Sca-1⁺ cells were small and co-expressed the endothelial cell marker PECAM (CD31⁺), but did not express c-kit, CD43, CD34 or CD8. Resident Sca-1⁺ cells express GATA4, MEF-2C and TEF-1, but did not express Nkx2.5 or genes encoding cardiac-restricted myofibrillar proteins. When cultures of cardiac-derived Sca-1⁺ cells were exposed to 5-azacytosine, approximately 2–4% of cells expressed sarcomeric proteins and exhibited striations. These cells, however, did not beat spontaneously or generate spontaneous electrical activity. Because 5-azacytosine may transform cells, other methods will have to be identified before Sca-1⁺ cells can be considered clinically. However, proof-of-concept studies revealed that Sca-1⁺ cells injected intravenously in an ischemia-reperfusion model of myocardial infarction home to the heart, engraft, but probably do not differentiate into cardiac myocytes. In fact, careful analyses revealed that most, if not all, of the injected Sca-1⁺ cells fused with resident myocytes.

More recently, Laugwitz and colleagues identified a small resident population of cells in the neonatal mouse, rat and human heart expressing Islet1 (Isl1⁺) that fulfill the criteria of a determined cardiac progenitor or cardioblast (77). Islet1 is a LIM-homeodomain transcription factor expressed in the anterior heart field (78). In the embryo, clusters of Isl1⁺ cells were observed in both atria, while in the ventricles they appeared as single cells. Of note, Isl1⁺ cells did not express c-kit demonstrating that they do not represent the same population of multipotent cardiac stem cells discussed above (75). Utilizing a novel conditional Cre-Lox fate mapping strategy, Laugwitz and colleagues made the surprising observation that 500–600 Isl1⁺ cardioblasts are detectable in the neonatal rat heart (77).

Isolated Isl1⁺ cells expressing GATA4 and Nkx2.5 may be expanded ex vivo. When co-cultured with neonatal cardiac myocytes, Isl1⁺ cells differentiate and express the full repertoire of cardiac myofibrillar genes, display spontaneous contractile activity and periodic calcium oscillations (77). Thus, in contrast to pleuripotent ES cells or multipotent lin⁻c-kit^POS stem cells, these Isl1⁺ cells appear to be determined cardiac myoblasts.

However, Isl1⁺ cells are exceedingly rare in the neonatal heart and may not exist in the adult heart. As such, the therapeutic potential of these cells relies upon the capacity to isolate these cells from the embryonic or neonatal heart and expand them ex vivo. However, these cells may represent an ideal cell system to define the molecular program regulating cardiac myocyte differentiation from cardiac myoblasts. Therefore, Isl1⁺ cells may serve as a useful intermediate between embryonic stem cells and mature cardiac myocytes to better define the steps in the developmental program regulating cardiac myocyte differentiation.

Conclusions

Stem cell therapeutics offer great promise for the treatment of myocardial infarction, cardiomyopathy and/or congestive heart failure. In less than a decade, we have moved from the first isolation of cells possessing the biological properties of cardiac stem cells or progenitors to phase I/II clinical trials assessing the safety and efficacy of bone marrow mononuclear cell infusions in the setting of acute myocardial infarction. Ultimately, it remains to be determined whether embryonic or adult stem cells will be successfully applied to regenerate functional myocardium in the clinical setting. Cardiovascular stem cell therapeutics is a field in its infancy and the premature application of cell therapies to patients without complete and rigorous characterization of these cells is dangerous and could ultimately slow down or halt progress in this promising field of research. It is critically important that we invest in basic science characterizing the biological properties and developmental potential of precisely characterized stem cell or cardiac progenitor cell populations. Further research is also required identifying factors in the diseased heart that promote regeneration or inhibit the process.

This research must proceed hand-in-hand with translational science directed toward optimization of stem cell delivery, engraftment and survival in the heart. At the same time, we must not lose sight of the principles that guide drug approval necessitating the careful analyses of dose-response relationships and potential systemic toxicity of cardiovascular cell therapies. Finally, we must learn from our recent experiences with angiogenic gene therapy trials (for review see (79)), and only perform well designed and controlled clinical trials with hard endpoints that will provide understanding of whether a particular therapy is efficacious and/or why it is not. If we proceed along this well trod pathway, the 21^st century may indeed usher in the era of regenerative medicine.

DISCUSSION

Sacher: Cincinnati: I enjoyed that very much. Since this seems to be, at least currently, a highly active area of interest, firstly, has anyone analyzed the cytokines that are likely to be liberated locally? And secondly, I noticed in your review of the literature that the majority of infusions involve bone marrow mononuclear cells. Why not consider mobilized peripheral blood-derived stem cells which would seem to me to be a good potential source of cytokines?

Parmacek: Philadelphia: Right. So I think most people think that delivery of regenerative cytokine (if it exists) would be a better way to go. The reason that bone marrow mononuclear cells were utilized in these early phase I clinical trials is that most groups were performing studies in the acute infarct setting. So they quickly obtained bone marrow aspirates and used a very simple purification scheme to isolate mononuclear cells which were injected down the coronary artery. These investigators didn’t have the time to grow up, expand or purify populations of stem or progenitor cells.

Boxer: Ann Arbor: I am concerned about the safety of injecting bone marrow cells down a coronary artery in the setting of acute myocardial infarction. Granulocytes would not be an issue, but I would worry about activated macrophages. And it would go back to Ron Sacher’s comment about being more critical in these studies, stressing the need to understand the biology of cells injected into the heart.

Parmacek: I agree 100%. One of the reasons I go around the country and present this overview of cardiac stem cells to primarily cardiologists is that there are many lessons to be learned from other disciplines. I hate to say it, but this kind of thing [jumping into clinical trials] has happened repeatedly in the recent history Cardiology. Currently, there are over 20 clinical trials on-going across the world, most of them without controls, delivering bone marrow mononuclear cells injected down coronary arteries with no data in animal models demonstrating long-term benefit or efficacy. There does appear to be a short term benefit in efficacy certainly in terms of functional indices. I think caution is very important, and that’s why I’ve started to give this talk.

Boxer: With the original publication I guess there was a lot of controversy. As you pointed out, there wasn’t repopulation of myocytes, but what may have occurred is fusion of bone marrow cells with myocytes.

Parmacek: I think that are several different explanations for the disparate results. I actually think that fusion occurs, but where it has been shown to occur, it occurs at such a low frequency that it does not explain these disparities, given the 60% repopulation numbers reported.