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EDITORIAL FOCUS
1Virginia Commonwealth University/Medical College of Virginia, Richmond, Virginia; and 2Yale University, New Haven, Connecticut
THE LOSS OF HEART MUSCLE during myocardial infarction is a major worldwide health issue, often leading to debilitating heart failure or death. Terminally differentiated cardiac muscle retains little if any capacity to undergo mitosis, and thus substantive regeneration of the heart after infarction does not occur. As such, mining the pluripotency of stem cells to regenerate lost heart muscle is an extremely attractive concept (6). A recent PubMed search for "heart and stem cell" yielded 
3,000 publications over the past five years, evidencing a remarkable investigational focus on cell therapy to rebuild damaged hearts. Many of these studies document significantly better cardiac function when cells are delivered to the heart after infarction (12, 21), and the field has rapidly moved to clinical testing (2, 15, 17, 27). Despite this widespread enthusiasm and hope, the evidence that stem cells delivered to the heart become mature contractile cardiac muscle is scant and controversial.
In the search for the "holy grail" of cardiac regeneration, several alternative paths have been taken. These include attempts to find the right marrow-derived cell population, use of cord blood-derived cells (10), use of cells derived from adipose tissue or other noncardiac somatic tissue source (4, 24), use of embryonic stem cells (11), attempts to identify progenitor cell niches in the heart (13, 19), and several alternative strategies to direct stem cell differentiation into the heart muscle. Following this last path, Orlandi et al. (20) report an elegant study of the fate of umbilical cord hematopoietic stem cells cocultured with cardiac myocytes (20). In their study, these investigators cocultured cord blood-derived human CD34+ cells with mouse cardiomyocytes. To track the fate of the human cells, cells were first transduced with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After a week in coculture, mononucleated EGFP+ cells underwent an extensive electrophysiological analysis that demonstrated multiple properties consistent with cardiac muscle differentiation, including action potential-associated cytosolic Ca2+ concentration ([Ca2+]i) transients, spontaneous sarcoplasmic reticulum [Ca2+]i oscillations, and membrane potential depolarization. These cells were also connected to surrounding cells by gap junctions.
Despite this functional and anatomical evidence of cardiac differentiation, RT-PCR of cocultured cells showed no expression of human-specific cardiac genes. There was, however, continued expression of human PGK1, providing evidence that the human cells remained viable and transcriptionally active. Thus, in this excellent well-controlled study, the authors convincingly demonstrate that functional measures alone are not sufficient to prove cardiac differentiation and, moreover, that physical association of CD34+ cells with cardiomyocytes does not induce full cardiac differentiation of these cells. These data strongly suggest that cardiac differentiation of stem cells is more than just a simple matter of association. The data do, however, suggest that physical contact between the CD34+ cells and cardiac myocytes is capable of inducing at least some phenotypic features of cardiac differentiation. Thus association seems to matter but is not a sufficient stimulus to impart full differentiation.
The data must, however, be interpreted in the context of the limitations of the study. As with all studies in which cardiac differentiation of stem cells purportedly occurs in association with fully differentiated cardiomyocytes, the possibility of cellular fusion must be considered. To exclude fusion, the authors fastidiously studied only mononuclear EGFP+ cells, the assumption being that cytoplasmic fusion would yield binucleate cells. As the authors acknowledge, this does not rule out nuclear fusion, which was not assessed. Nonetheless, even if the assumption of nuclear fusion is made, other questions arise. What determines which genes will be expressed within fused nuclei? Why did the constitutive expression of a noncardiac human gene continue, whereas no expression of human cardiac genes was observed? These questions are representative of the myriad of questions still unanswered in the field of regenerative medicine and reflect the immense complexity of the biology involved.
Among the unanswered questions are those of how cell-cell contact and the cellular microenvironment contribute to cell fate determination. Stem cells express integrins and other transmembrane proteins with extracellular domains, and these may play an essential role in transducing signals from the microenvironment into fate-determining biological events in the stem cells (3, 14, 16, 30). Cell-cell contact with stromal cells has been shown to induce marked alterations in gene expression in stem cells and to alter their proliferation (29). Paracrine signals in the microenvironment likely also play a critical role in determining cell fate (8), as may specific physical parameters such as oxygen tension (1). In addition to all of these considerations, it must be remembered that although they are distinctly different cell types, the DNA instruction set within a cardiac muscle cell is the same as is found in a retinal rod, or a Purkinje cell in the cerebellum. Much of the information that defines which genes will be expressed in each of these unique cell types is contained in epigenetic determinants such as DNA methylation patterns and histone acetylation (5, 23). These epigenetic determinants of cell fate are still poorly understood (25, 26, 28), and in a very real sense the same biological complexities that have vexed cancer biologists trying to understand malignant transformation are confronting the regenerative medicine biologists trying to push cells toward specific differentiation pathways.
Still, among all this complexity, progress is being made. In an intriguing attempt to provide the optimal environment for the generation of heart muscle from cells, a recent report described the use of decellularized hearts. In the reported study (22), rat hearts were decellularized by treatment with detergents, leaving essentially only the extracellular matrix scaffolding behind. When endothelial cells and neonatal cardiac myocytes were introduced into these decellularized hearts, followed by incubation in a bioreactor, they organized into an endothelialized vasculature and enough cardiac muscle to allow cardiac contraction and force generation. Although this study did not use stem cells, the possibility of using this type of advanced tissue scaffolding in conjunction with pluripotent cells is an exciting consideration.
The current study by Orlandi et al. (20) is significant because it very elegantly demonstrates that the "differentiation by association" paradigm is oversimplistic. Ultimately, studies such as theirs clarify that there is still much science to be done, and this will help advance the field. As a closing note, even though we currently do not appear to know enough to push cellular differentiation down the pathways we chose, cell therapy may still be beneficial in the heart by other mechanisms. Cardiac myocytes are a major source of autocrine and paracrine factors produced in the heart (7), and this capacity can be markedly diminished by the loss of heart muscle during infarction. Interestingly, it may actually be the repletion of these paracrine factors by cells delivered to the heart after infarction that is primarily responsible for the beneficial effects that have been observed (9, 18).
FOOTNOTES
Address for reprint requests and other correspondence: F. J. Giordano, Center for Vascular Biology and Transplantation, Yale School of Medicine, 10 Amistad St., Rm. 426, New Haven, CT 06520 (e-mail: frank.giordano{at}yale.edu)
REFERENCES
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