Reviews. This Review is part of a thematic series on Cellular Therapy, which includes the following articles:

Reviews This Review is part of a thematic series on Cellular Therapy, which includes the following articles: The Stem Cell Movement Genetic Enhancement of Stem Cell Engraftment, Survival, and Efficacy Disease and Aging As Modifiers of Efficacy Paracrine Signaling in Cell Transplantation Assessment and Optimization of Cell Engraftment After Transplantation Immune Biology of Stem Cells Cardiogenic Differentiation and Transdifferentiation of Stem Cells Stem Cell Homing to Sites of Injury Regulatory Considerations in Cell Transplantation Eduardo Marbán, Editor The Stem Cell Movement Nicola Smart, Paul R. Riley Abstract Stem or progenitor cell based strategies to combat ischemic heart disease and myocardial infarction, whether autologous transplantation or stimulation of resident populations, not only require detailed insight into transdifferentiation potential and functional coupling, but the efficacy of this approach is underpinned by the need to induce appropriate migration and homing to the site of injury. This review focuses on existing insights into the trafficking of stem cells in the context of cardiac regenerative therapy, with particular focus on the wide variety of potential sources of cells, critical factors that may regulate their migration, and how extrapolating from embryonic stem/progenitor cell behavior during cardiogenesis may reveal pathways implicit in the adult heart postinjury. (Circ Res. 2008;102:1155-1168.) Key Words: cardiac stem cells migration homing mobilization Regulated migration of stem cells critically underlies organogenesis during development and in homeostasis and repair in the adult. Achieving targeted trafficking of stem cells is critical for effective tissue regeneration by inducing mobilization, either from an endogenous niche or following systemic administration. Furthermore, the ability to manipulate stem cell homing means that these cells offer themselves as vehicles for in vivo delivery of therapeutic genes or drugs. Stem cells perform important functions in the establishment of embryonic tissues during development and, in some cases, are retained into adulthood where they support homeostasis through the continued replacement of senescent cells and regeneration of injured or diseased organs. The regenerative capacity of the human myocardium is, however, grossly inadequate to compensate for the severe loss of viable heart muscle that follows myocardial infarction (MI). Existing therapies for heart failure, the leading cause of death worldwide, only serve to delay progression of the disease. More recent approaches have focused on replacement of injured myocardium with healthy cardiomyocytes, and the induction of neovascularization and significant effort has been invested in the search for the optimal embryonic or adult progenitor cells with which to replace damaged cells. The progress made in identification and understanding of novel stem/progenitor cells has been tempered by the variable and controversial results of translational and clinical studies. 1,2 A paradigm has emerged which uses an understanding of the molecular programming of cardiovascular progenitors that build the developing heart for extrapolation to repair of the injured adult heart. Cardiogenesis requires extensive Original received March 5, 2008; revision received March 31, 2008; accepted April 4, 2008. From the University College London-Institute of Child Health, United Kingdom. Correspondence to Dr Paul R. Riley, Molecular Medicine Unit, UCL-Institute of Child Health, 30 Guilford St, London WC1N 1EH, United Kingdom. E-mail P.Riley@ich.ucl.ac.uk 2008 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.108.175158 1155

1156 Circulation Research May 23, 2008 specification, expansion and differentiation of cardiac and vascular progenitors, a property believed to be restricted to the embryonic heart. However, the recent identification of resident progenitor populations in the adult heart offers new therapeutic targets for myocardial repair and neovascularization. The angiogenic potential of vascular tissues emerges during organ growth and remains dormant within the postnatal, steady-state organism until elicited again during repair, regeneration, or neoplasia. It is now becoming apparent that regeneration is underpinned by a reactivation of the embryonic program that created the tissues in the first instance; as such, there is much to gain from understanding the embryonic mechanisms of vasculogenesis and cardiomyogenesis and developing appropriate strategies to redeploy these pathways for therapeutic benefit. The self-renewal and transdifferentiation capacities of stem cells are worthless unless their migration to target tissues can be appropriately orchestrated. For many of the stem/progenitor cells currently under investigation the molecular and cellular mechanisms underlying their migration are not fully understood. However, novel insights may be derived from investigating the trafficking mechanisms of the closely related hematopoietic cells or from an understanding of progenitor migration during embryogenesis, and this review incorporates examples from these 2 settings. Cell-Based Cardiac Regeneration Therapy: Which Cell Types? Skeletal Myoblasts Autologous skeletal myoblasts were among the first cell types tested in the context of cardiac regeneration, 3 an obvious choice given their resistance to ischemia and ability to regenerate after injury. 4 They form myotubes in vivo but appear unable to differentiate into cardiomyocytes and yet are reported to improve ventricular function in animal studies. 3 Human trials are ongoing, although, in some, lack of efficacy has resulted in their premature termination. 5 The main factor limiting the therapeutic use of skeletal myoblasts is their failure to integrate electrically with surviving cardiomyocytes, 6 posing a greater risk of arrhythmia. Furthermore, skeletal myoblasts do not extravasate (transverse the vascular endothelium) and migrate to ischemic areas 7 and may even obstruct distal microcirculation after intracoronary administration, leading to embolic myocardial damage. 8 Embryonic Stem Cells As the prototypical stem cell, embryonic stem (ES) cells unequivocally fulfill all of the criteria of stemness: clonality, self-renewal, and multipotency. 9 ES cells can differentiate into all cell types required in the adult and hold the potential to completely regenerate the myocardium. Ethical issues, technical hurdles with maintaining survival of transplanted cells, and the concerns of immunologic rejection and teratoma formation 10 stand as obstacles in the path of ES cell based therapy. However, these limitations may be overcome with advances in our understanding of the specification and differentiation of ES cell derived cardiomyocytes, alongside methods to limit tumorigenesis, including genetic preprogramming or in vitro differentiation before injection. 11 Three recent studies used developmental approaches to identify multipotent cardiovascular progenitor cells in differentiating mouse ES cell cultures 12 14 and identified Nkx2 5/ Isl1/Flk1 as the transcriptional signature of such progenitors. Cardiomyocytes derived from mouse and human ES cells have been transplanted to form stable grafts in the uninjured hearts of mice, rats, and pigs, 15 18 resulting in improved contractile function, 19 with no evidence of arrhythmias. Importantly, human (h)es cell derived cardiomyocytes continued to proliferate within the graft, making it possible to use small implants that will expand over time to replace injured tissue. Protocols to enhance the cardiomyocyte potential of ES cells, along with the development of a survival cocktail to improve postgraft survival, have enabled significant cardiac regeneration using ES cells. 20 The degree of long-term regeneration using ES cells is still debatable, and the extent and mechanism of their migration within the heart following transplantation has not been determined. Much ongoing work focuses on influencing cardiomyocyte specification from ES cells by manipulating signaling pathways, such as the Wnt/ -catenin and bone morphogenetic protein pathways, to enable the therapeutic strategy of preselecting committed ES derived cardiac progenitors for transplantation. 21 Induced Pluripotent Stem Cells Cardiovascular cells derived from human ES cell sources are highly desirable for cardiac therapy but will continue to be hampered by ethical constraints. In this regard, an exciting recent development was the discovery that human dermal fibroblasts can be reprogrammed to pluripotency by ectopic expression of 4 transcription factors (Oct4, Sox2, Klf, and Myc). 22 Induced pluripotent stem (ips) cells resemble ES cells in morphology, gene expression profile, differentiation potential, teratoma formation, and ability to generate germline chimeric mice. More recent studies have revealed an enhanced selection of ips cells based on Nanog expression and demonstrated their induction in the absence of the myc protooncogene, 23 further increasing their potential for therapeutic use. Although these cells have yet to be tested in cardiac transplantation they may 1 day be the cell of choice, offering the additional benefit of individual patient-specific cells. Multipotent Adult Germline Stem Cells Adult spermatogonial stem cells were recently isolated from adult mouse testis and shown to acquire certain ES cell properties, 24 including multipotency and germline transmission. These so-called multipotent adult germline stem cells (magscs) efficiently differentiate into ventricle-, atrial-, pacemaker-, and Purkinje-like cardiomyocytes, which exhibit rhythmic Ca 2 transients and beating. 25 Transplanted magscs were able to proliferate and differentiate in normal murine hearts, suggesting that magscs could provide a source of suitable cardiomyocytes for potential therapeutic application.

Smart and Riley Stem Cell Migration 1157 Bone Marrow Derived Cells A pioneering study in mice suggested that bone marrowderived hematopoietic stem cells could differentiate into cardiomyocytes and that their introduction into remote myocardium following MI could induce repair of injured muscle. 26 However, subsequent studies with genetically labeled cells have indicated that bone marrow derived stem cells (BMSCs) do not transdifferentiate into cardiomyocytes. 1,27 29 The overall conclusion from a number of such studies is that although very few, if any, bone marrow derived cells integrate into the myocardium, an appreciable improvement in myocardial function was demonstrated and this was attributable to the cardioprotective or proangiogenic effects of secreted paracrine factors. Despite the controversy concerning the efficacy of BMSCs, a number of large scale multicenter clinical trials have been initiated in which autologous progenitor cells derived from bone marrow were reintroduced, following in vitro expansion, via intracoronary infusion into patients with acute myocardial infarction (AMI). 30 To date, the majority of clinical studies have used bone marrow mononuclear cells and have shown, at best, modest improvements in cardiac function. 2 The majority of trials conclude that any benefits conferred likely result from the paracrine secretion of cytokines and growth factors and the resulting angiogenesis and improved microvascular function, as demonstrated in the REPAIR-AMI study. 2 Equally controversial is the degree of BMSC homing reported in various studies. 31,32 Given the general consensus that the benefits of BMSC therapy derive primarily from secretion of paracrine factors, appropriate homing of these cells is essential to enable such factors to function, because their effects are, by definition, limited to a short range, requiring relatively high local concentrations. Endothelial Progenitor Cells Endothelial progenitor cells (EPCs) are a specialized subset of hematopoietic cells found in the bone marrow and peripheral circulation. 33 During development, endothelial and hematopoietic cells arise from a common progenitor, the hemangioblast, a cell type considered to be restricted to the embryo. However, as demonstrated by Asahara et al, up to 20% of the CD34 population of peripheral blood in the adult are also vascular endothelial factor receptor (VEGFR) and phenotypically characterized by antigens usually associated with hematopoietic stem cells, including CD133, CD34, c-kit, VEGFR2, CD144 (cadherin), and Sca-1. 33 Until recently, new blood vessel growth in the adult was thought to occur exclusively by angiogenesis (sprouting from existing vessels), but the discovery of circulating EPCs permanently changed this view. Vasculogenesis in the developing embryo is retained and redeployed when required in the adult. EPCs are mobilized from bone marrow and recruited to foci of neovascularization, where they from new blood vessels in situ. Striking parallels in the regulatory steps of embryonic and postnatal vasculogenesis suggest that the underlying initiating stimuli and regulatory pathways may be conserved. EPCs are incorporated into injured vessels and develop into mature endothelial cells during the processes of reendothelialization and neovascularization. 34 On differentiation, CD133 expression is lost and EPCs begin to express vascular endothelial cadherin and von Willebrand factor. 35 EPCs have not been shown to differentiate into cardiomyocytes but appear to promote angiogenesis 36 and likely provide paracrine survival signals to cardiomyocytes. 37 Angiogenic growth factors including VEGF-A, VEGF-B, stromal cell derived factor (SDF)-1, and insulin-like growth factor-1 are themselves secreted at high levels from EPCs, and these elicit a potent migratory response on mature endothelial cells and cardiac resident c-kit progenitor cells. 38 Thus, in addition to the physical contribution of EPCs to newly formed vessels, an equal, if not greater, mode of EPC action may, as with BMSCs, be the paracrine secretion of proangiogenic factors. The use of EPC populations for therapeutic purposes has rapidly progressed into clinical trials with promising preliminary results. 39,40 Very Small Embryonic Like Stem Cells The bone marrow also harbors an extremely rare (1 per 10 4 to 10 5 BM-MNCs) population of very small embryonic like (VSEL) cells, identified by virtue of their chemotactic response to SDF-1. 41 VSEL cells express the stem cell markers stage-specific embryonic antigen, Oct-4 and Nanog as well as the SDF-1 receptor, CXCR4. 42 These highly motile cells are hypothesized to have been deposited during development and remain as circulating pluripotent stem cells that play an important role in tissue homeostasis. Two factors, relating to the differentiation capacity of VSELs, may limit their therapeutic potential; firstly, the ability to form VSEL-derived spheres (VSEL-DS) or to expand into cells from all 3 germ layers is a phenomenon which cannot be reproduced with human VSEL cells 42 ; secondly, VSEL-DS formation, at least in mice, appears to be restricted to cells from younger animals but lost in old ( 2-year-old) mice. 42 Endogenous Cardiac Stem Cells Failure of BMSCs (including EPCs) thus far to deliver the holy grail of myocardial regeneration has led researchers to focus on the identification of resident cardiovascular stem/ progenitor cells and factors that stimulate their proliferation or migration. The adult heart was for many years believed to be a postmitotic organ comprising only fully differentiated cells, which need to survive a lifetime without the possibility of support from newly formed vascular or myocardial cells. The first indication that the adult heart harbored a population of stem cells possessing regenerative properties came from a study of AMI patients showing an elevated number of immature cardiomyocytes, capable of mitotic division, in the infarct border zone. 43 Whether these progenitors were of endogenous or circulating origin was uncertain, but the identification of a resident stem cell population within the heart, which supports myocardial regeneration, has exposed new opportunities for cardiac repair. 44 Two approaches are currently under investigation: the first is to explant cardiac stem cells from the heart, induce their proliferation and differentiation ex vivo and to engraft them back into the damaged heart 45 ; and the second, which to date has received less attention, is to stimulate cardiac progenitor cells (CPCs)

1158 Circulation Research May 23, 2008 Table. Phenotypic and Migratory Characteristics of Stem Cells for Cardiac Repair Stem/Progenitor Cell Type Phenotypic Markers Differentiation Potential Frequency in Adult Heart Mode of Delivery Stimulatory Factors Skeletal myoblasts Myf5/MyoD; Wnt Myotubes NA Direct injection to ventricle NA 5a/Wnt 5b wall ES Oct4, Nanog Pluripotent NA Direct injection/i.c. NA ips Oct 4, Sox2, Klf, Pluripotent NA? Direct/i.c. (Myc), (Nanog) magcss ES cell markers Multipotent NA? Direct injection/i.c. NA BMSCs c-kit, Sca-1 Multipotent Some c-kit CPCs may be derived from BM; mobilized upon stimulation VSEL cells SSEA, Oct4, Nanog, CXCR4 EPCs CD34, CD133 c-kit, VEGFR2, CD144, Sca-1 in situ to proliferate, migrate, and differentiate in the infarcted heart without ex vivo manipulation. To this end, a major therapeutic goal is the identification of factors that stimulate cardiac stem cells to form replacement cardiomyocytes and vascular progenitors for regeneration of the injured heart. The adult myocardium contains a small fraction of stem cells that express the cell surface markers c-kit 44 and Sca1. 46 Side-population (SP) cells, characterized by their ability to exclude Hoechst dye, were first identified in bone marrow but have subsequently been found in the heart. 47 The SP cell population includes some cells that express c-kit and/or Sca1 and, like c-kit /Sca1 cells, can give rise to cardiomyocytes. Cardiospheres, derived from human percutaneous endomyocardial biopsy specimens, yield cardiosphere-derived cells with the potential for cardiogenic differentiation. 48 On injection into the border zone, cardiosphere-derived cells migrated into the infarct zone and improved the proportion of viable myocardium and cardiac function. One of the most promising populations of endogenous cardiac progenitor cells are the recently discovered multipotent Isl1 cardiovascular progenitors (MICPs) because they have potential to form cardiac, smooth muscle, and endothelial cell lineages. 13,49 However, Isl1 cells, like the c-kit / Sca1 populations, are exceedingly rare in the neonatal heart (500 to 600 per rat heart) and may not even exist in the adult heart. The therapeutic potential of these cells may, therefore, depend on the ability to isolate them from the embryonic or postnatal heart and expand them ex vivo. 50 i.c. following ex vivo expansion or stimulate homing G-CSF, SDF-1, VEGF, EPO, SCF, MIP-2, GRO-, IL-8 Pluripotent NA Stimulate homing NA ECs Mobilized from BM upon stimulation i.c. following ex vivo expansion or stimulate homing CPCs c-kit and/or Sca-1 CMs Rare resident population i.c. following ex vivo expansion or stimulate in situ CDCs MICPs EPDCs c-kit, CD105, CD90 CD34, CD31 Isl1/Nkx2 5/Flk- 1CMs, Epicardin, Tbx18 RALDH2, WT-1 CMs, ECs SMCs, ECs conduction cells Fibroblasts SMCs, ECs Derived from rare CPC population Rare in postnatal; not shown in adult Rare but induced to migrate and proliferate Direct injection Potential for i.c. following expansion Stimulate in situ G-CSF, SDF-1, VEGF, EPO, enos, EPO, Ang-1, PDGF HMGB-1 Unknown Unknown ips indicates induced pluripotent cells; CDCs, cardiosphere-derived cells; MICPs; ECs, endothelial cells; SMCs, smooth muscle cells; CMs, cardiomyocytes; BM, bone marrow; i.c., intracoronary; EPO, erythropoietin; MIP-2, macrophage inflammatory protein-2; GRO-, growth regulated oncogene- ; Ang 1, angiopoietin-1; enos, endothelial NO synthase; PDGF, platelet-derived growth factor. Migration of MICPs has yet to be explored. Isl1 cardiac cells do not express c-kit or Sca-1 and may not, therefore, use the same pathways for homing to infarcted tissue as the classic stem cells. The only insight into MICP migration derives from our understanding of how Isl1 progenitors migrate during cardiac development. 51 Isl1 is required for the survival, proliferation, and migration of embryonic myocardial precursors, in other words, for maintaining their stemness. Isl1 cells do not express markers of mature cardiomyocytes, and Isl1 transcription is switched off as precursor cells differentiate, leading to the hypothesis that Isl1 function is required for cell expansion and migration into the forming heart. More recently, the potential of epicardium-derived progenitor cells (EPDCs) for regeneration of the adult heart has been recognized. 52,53 Another example of the paradigm of recapitulating embryonic roles for therapeutic application is that of the epicardium and its derivatives, which play an essential role in formation of the coronary vasculature during development and appear to retain the same proangiogenic potential in the adult. 52 Epicardial cells, originating from the embryonic proepicardium, undergo epithelial-to-mesenchymal transformation (EMT) and possess extensive migratory capacities, on stimulation by paracrine factors from the underlying myocardium. 54 However, unlike in the embryo, the adult epicardium lies quiescent and requires stimulation by those factors that promote coronary vasculogenesis during development. To date, the only identified factor for stimulating adult EPDCs is the actin-binding protein thymosin T 4

Smart and Riley Stem Cell Migration 1159 Bone Marrow Mobilization Ischemic myocardium Epicardium Cardiomyocyte BMSC/EPC Resident Cardiac Progenitor Cell (CPC) CPC-derived cardiomyocyte low O 2 SDF-1 endogenous CPC niche (T )4. 52 EPDCs, both in embryonic and adult heart, are multipotent and differentiate into at least 3 cell types: fibroblasts, endothelial cells, and smooth muscle cells. Significantly, recent reports indicate that epicardial cells may even form cardiomyocytes, thereby elevating their status to that of a cardiac stem cell 55 with potential for repair. This review focuses primarily on the mechanisms that regulate the migration of stem cells. Although a clear picture has emerged of the processes adopted by BMSCs, including EPCs, for migration (mobilization and homing), for the more recently identified endogenous cardiac progenitors, little is known about their migration in the adult heart. However, recent studies reveal that the mechanisms that promote migration of such progenitors during development can also be reactivated in the adult heart if appropriately stimulated, and regulators of progenitor cell migration are now recognized as potential therapeutic targets for cardiac regeneration. Mechanisms of Stem Cell Migration In this section, we address the migratory mechanisms used by the classic (c-kit ) stem cell types and endogenous cardiac progenitors (summarized in the Table and Figure). Although examples are given primarily for bone marrow hematopoietic stem cells (HSCs) and EPCs, it appears likely Proliferation Chemokine e.g. SDF-1 Myocardial Tβ4 Exogenous Tβ4 Tβ4 receptor migration + differentiation Paracrine factor e.g.hmgb1 EPDC Endothelial cell Smooth muscle cell Figure. Stem cell migration for cardiac therapy. Bone marrow stem cells and resident cardiac progenitor cells reside within permissive niches until stimulated by hypoxia (following myocardial ischemia) or paracrine factors, such as HMGB1 or thymosin 4. The identification of factors which stimulate resident cardiac stem or progenitors cells to induce cardioprotection, initiate vascular growth and promote myocardial repair could lead to the development of therapeutic interventions for ischemic heart disease. that many types of stem cell share common mechanisms for mobilization and homing. 56 Much of our knowledge of stem cell migration derives from our understanding of the mechanisms that enable HSCs to leave the bone marrow and enter the circulation, relocate to a distant tissue, and return to the bone marrow. Present research aims at directing the targeted migration of stem cells toward ischemic tissues for regeneration and emerging evidence implicates the same mechanisms in homing to target organs as to bone marrow. Hematopoietic Stem Cell Recruitment and Homing During embryogenesis, blood-forming stem cells migrate from the fetal liver via the circulation, home to the bone marrow, and repopulate it with high numbers of immature and maturing blood cells of all lineages. These, in turn, are released into the circulation while maintaining a small pool of undifferentiated stem cells within the bone marrow. 57 HSC recruitment (mobilization) and homing are mirror processes regulated by the interplay of cytokines, chemokines, and proteases. 58 Essentially, HSC recruitment is characterized both by loss of cell cell contacts (via downregulation of cell adhesion molecules) and a desensitization of chemokine signaling, notably the SDF-1 /CXCR4 axis, the fundamental

1160 Circulation Research May 23, 2008 signaling pathway underlying stem cell mobilization and homing during homeostasis and injury. 59 Conversely, stem cell homing requires upregulation of cell adhesion molecules and activation of the SDF-1 /CXCR4 axis. The regulation of the SDF-1 /CXCR4 axis in relation to stem cell migration is discussed below. Chemokine Signaling Chemokines are defined as small peptides that initiate the migration of effector cells. Although mobilization of HSCs by cytokines requires 5 to 6 days to attain peak response, chemokines induce mobilization within 30 minutes to a few hours. Mobilization of HSCs from bone marrow is achieved by the action of cytokines, such as granulocyte colonystimulating factor (G-CSF) 60 and the closely related granulocyte/macrophage colony-stimulating factor (GM-CSF), 61 Flt-3 ligand, 62 erythropoietin, 63 and stem cell factor (SCF) (the ligand for c-kit) 64 ; growth factors such as vascular endothelial growth factor (VEGF), 65 angiopoietin-1, 66 and placental growth factor, 67 as well as several chemokines such as SDF-1, 68 interleukin (IL)-8, 69 growth-regulated oncogene-, 70 and macrophage inflammatory protein-2. 71 The first suggestion that cytokine-induced stem cell mobilization may be used to enhance cardiac repair came from studies to increase EPC levels for neovascularization in hind limb ischemia. VEGF 72 and GM-CSF 73 were found to augment EPC levels and improve neovascularization. Hematopoietic stem cell mobilizing factors G-CSF and SCF were subsequently shown to improve cardiac regeneration in mice, 31 and this and other small scale animal studies rapidly led to initiation of clinical trials to assess the ability of G-CSF to mobilize stem/progenitor cells in patients with coronary artery disease 74 and AMI. 75 G(M)-CSF mobilized blood from patients contained 5- to 100-fold higher levels of HSCs, MSCs, and EPCs, compared with nonmobilized blood 73 75 ; however, the ability of these cells to improve cardiac remodeling and function after AMI has been disappointing. 76 Although the idea that recruited BMSCs differentiate into cardiomyocytes to any significant degree is now generally discounted, G-CSF treatment has been shown to induce angiogenesis in the infarcted heart and to have a paracrine protective effect on cardiomyocytes. 76 Chemokine signaling for directing migration is an embryonic principle because directed cell movement is a fundamental requirement for tissue formation. Chemokine receptors, predominantly CXCR4, have been detected from embryonic day (E)7.5, coexisting spatially and temporally with SDF-1, and have been implicated in the organogenesis of cardiovascular, neuronal, hematopoietic, and craniofacial systems. 77 Once organogenesis is complete, the progenitor pool is diminished and chemokine signaling switches to promote maintenance within stem cell niches over active tissue recruitment. However, the progenitors remain available but dormant such that tissue injury, leading to chemokine induction, can promote redeployment of progenitor cells to mediate regeneration. Additional repair, in the form of continuous low-level progenitor cell recruitment, provides a mechanism for tissue homeostasis. The SDF-1/CXCR4 Axis: A Central Regulator of Stem Cell Migration SDF-1 represents the major chemokine for initiating stem cell migration. 78 The majority of cytokines that mediate stem cell migration do so via modulation either of SDF-1 or of its receptor, CXCR4. Thus the SDF-1/CXCR4 axis is central for stem cell mobilization from the bone marrow and homing to ischemic tissues. 79 SDF-1 is constitutively expressed on bone marrow endothelium, leading to integrin-mediated arrest of stem cells despite a prevailing shear stress. 80 Under homeostatic conditions, most progenitors remain in the bone marrow compartment, retained by high SDF-1 expression, which is maintained by the hypoxic microenvironment. It is possible that low numbers of progenitor cells continuously recirculate under physiological conditions. However, after an ischemic insult, soluble factors such as SDF-1 are released by injured tissue and stimulate mobilization of progenitor cells from the bone marrow. These cells are recruited to ischemic tissue by high local levels of SDF-1, providing a permissive niche. Following MI, SDF-1 gene therapy was shown to enhance HSC recruitment to the heart, offering a promising therapeutic strategy for mobilizing stem cells to ischemic myocardium. 81 EPC mobilization from the bone marrow was shown to be induced by SDF-1, 82 VEGF, 82 angiopoetin-1, 66 erythropoietin, 63 G-CSF, 83 GM-CSF, 73 endothelial NO synthase, 84 estrogen, and platelet-derived growth factor. 85 The principal physiological stimulus behind EPC mobilization is tissue hypoxia, which results in increased cytokine production. SDF-1 expression is directly proportional to reduced local tissue oxygen tension, 79 and endothelial expression of SDF-1 is believed to act as a danger signal to indicate the presence of tissue hypoxia. Two different physiological environments in which local tissue oxygen tension is decreased and progenitor cell recruitment occurs are within the bone marrow and during embryonic development. In the bone marrow, the normal physiological oxygen tension is hypoxic, 79 and SDF-1 is constitutively expressed, providing a robust and wellcharacterized niche for stem and progenitor cell recruitment. During embryonic development, rapid tissue growth outpaces the developing vascular system, creating areas of local hypoxia with SDF-1 expression, and is believed to guide primordial stem cells to form new vascular networks by vasculogenesis. 77 Moreover, this sequence was also demonstrated to occur in the postnatal organism under pathophysiological conditions, with endothelial SDF-1 selectively upregulated in transiently ischemic tissue, guiding the recruitment of progenitor cells to a permissive niche where they proliferate and assemble. 86 These data suggest that the endothelium functions as an active participant in postnatal tissue repair in much the same way that nascent vascular networks regulate organogenesis in the developing embryo. 87 Ceradini et al identified a hypoxia-responsive element containing binding sites for hypoxia-inducible factor 1 in the SDF-1 promoter and showed that this element was critical for hypoxia-induced SDF-1 expression. 79 Functionally, hypoxiainducible factor 1 induced SDF-1 expression facilitated adhesion of progenitor cells to ischemic endothelium and

Smart and Riley Stem Cell Migration 1161 induced their migration in transwell assays, suggesting a specific role in homing of circulating progenitor cells to ischemic vascular beds. Blockade of either CXCR4 on infused progenitor cells or SDF-1 in the host resulted in a dramatic reduction in progenitor cell recruitment to ischemic tissue and decreased the resultant neovascularization and blood flow. 79 These findings suggest a requirement for cooperation between a combination of factors that regulate homing (chemokines, selectins, and integrins) and engraftment (matrix metalloproteases and cathepsins) to determine the specific cell types recruited to ischemic tissue. Cytokine Regulation of the SDF-1/CXCR4 Axis The expression of CXCR4 on stem cells is very dynamic and its regulation by cytokines and chemokines is well documented. Flt3-ligand, 62 SCF, 88 IL-3, IL-6, and IL-8, 89 hepatocyte growth factor, 90 and G-CSF 91 impact directly on the SDF/CXCR4 pathway. G-CSF induced cell migration depends both on disruption of cell cell contacts by cleavage of cell adhesion molecules and a negative effect on CXCR4/ SDF-1 signaling by dual inactivation of both the receptor and ligand. Following G-CSF administration, active neutrophil proteases such as elastase and cathepsin G accumulate within the bone marrow, leading to downregulation of the SCF receptor and a dramatic reduction of vascular cell adhesion molecule (VCAM)-1 expression in the bone marrow. 92 Concomitant with changes in protease levels, cleavage at the N-terminus of CXCR4 on HSCs, results in their mobilization from bone marrow into the peripheral circulation. The SDF-1/CXCR4 signal is also inactivated at the level of the ligand by cleavage of SDF-1 by dipeptidylpeptidase IV. 93 IL-8 has been proposed to use matrix metalloprotease (MMP)-9 activity, instead of elastase and cathepsin G, to induce mobilization, although how exactly this impacts on SDF-1/CXCR4 is not fully understood. Flt3-ligand, in combination with G-CSF, induces BMSC mobilization in mice, resulting in improved cardiac repair, which is significantly greater than that achieved with G-CSF alone. 94 Whereas short-term exposure to Flt3-ligand was shown to enhance SDF-1 induced migration, long-term exposure resulted in downregulation of CXCR4, 62 suggesting an extrinsic regulatory role for Flt3-ligand in HSC migration with implications for homing to the bone marrow as well as mobilization. Clinical studies to enhance the mobilization efficacy of HSCs therapeutically led to trials involving the aforementioned chemokines, either alone or in combination, many of which show promise for their use in inducing stem cell mobilization eg, a truncated form of growth-regulated oncogene- with G-CSF 95 and AMD3100 (a CXCR4 antagonist) with G-CSF. 96 The recent discovery that CXCR7 is also a receptor for SDF-1 97 changed the notion of a monogamous relationship between the chemokine and its first identified receptor, CXCR4. However, whether CXCR7 has a role to play in stem cell migration remains equivocal. Balabanian et al showed that CXCR7 activation promotes migration of T cells, 97 whereas a subsequent study argues that the receptor plays no role in migration. 98 Boldajipour et al have perhaps reconciled some of these discrepancies and demonstrate a role for CXCR7 in chemokine-guided cell migration, 99 albeit indirectly. The authors showed that CXCR7 functions to internalize SDF-1, thereby maintaining a sink for the chemokine. Not only does this study reveal the importance of ligand clearing in directed cell migration, by regulation of chemokine signaling at the protein level in addition to known transcriptional regulation, but it also indicates cooperation between chemokine receptors to regulate cell migration. It has recently been shown that circulating HSCs and their progenitors exhibit robust circadian fluctuations in antiphase with the expression of the chemokine SDF-1 in bone marrow, which is regulated via circadian noradrenalin secretion by the sympathetic nervous system. 100 These data demonstrate a circadian release of circulating stem cells during the resting period that may constitute an essential component of the homeostatic regeneration program, either of the bone marrow niche itself or of damaged tissues. Stem Cell Homing The term homing describes the migration of a circulating stem cell into a target tissue or the bone marrow. Homing constitutes a multistep cascade comprising: recognition and interaction with microvascular endothelium, transmigration through the endothelium, and, finally, migration and invasion of the target tissue, a process that relies on a complex interplay between cytokines, chemokines, adhesion molecules, and extracellular matrix degrading proteases. The capacity of stem cells to migrate and invade is critical for functional integration even when cells are injected directly into the site of injury. Although the mechanisms of progenitor cell homing to sites of tissue injury are poorly understood, some insight can be gained from parallels with the homing of hematopoietic progenitor cells to bone marrow. 101 HSCs are required to transmigrate across the vascular endothelium, a process known as extravasation. Although recent studies have offered insight into the mechanisms of extravasation, much of the detail remains imprecise. Galactose and mannose specific lectins and their receptors have been implicated in successful bone marrow engraftment 102 and the rolling phase of extravasation is mediated by E- and P-selectins. 103,104 Subsequent firm attachment is mediated via intercellular adhesion molecule-1/leukocyte function associated antigen-1, VCAM-1/very late antigen (VLA)-4 ligand pairs, and junctional adhesion molecules, such as platelet endothelial cell adhesion molecule. 103 Such interactions have been shown to be critically important because HSC homing was inhibited by at least 90% by blocking antibodies to VLA-4 ( 4 1 integrin) or VCAM-1. 105 Integrins mediate the adhesion and transmigration of stem and progenitor cells. In particular, 2 integrins were shown to be essential for homing and improvement of neovascularization mediated by EPCs after hindlimb ischemia. 106 Integrin-dependent adhesion of EPCs is another aspect of cell migration regulated by SDF-1. 107 As with stem cell mobilization, a crucial factor in regulating homing is SDF-1, which was shown to trigger the firm adhesion of leukocyte function associated antigen-1 to intercellular adhesion molecule and VLA-4 to VCAM-1 under

1162 Circulation Research May 23, 2008 shear flow conditions. 108 Both VLA-4 and VLA-5 are required for SDF-1 induced polarization and extravasation of HSCs through the extracellular matrix underlying the endothelium 80 ; polarization is fundamentally required for cell migration. Other cytokines, including GM-CSF, IL-3, and SCF, trigger the adhesion of HSCs during homing by activating the 1 integrins VLA-4 and VLA-5. Transendothelial migration of cells requires degradation of basement membrane, which is dependent on the production of matrix-degrading enzymes, especially those capable of degrading type IV collagen, such as MMPs. 109 MMP-2 and MMP-9 are known to be expressed by all cell types that migrate through the endothelial cell layer including leukocytes, 110 tumor cells, 111 and HSCs. 112 Homing of Stem Cells to the Heart What are the factors that control endogenous trafficking of progenitor cells to areas where they are needed for repair? BMSCs or cardiac progenitors expanded ex vivo and reintroduced systemically home to an infarcted heart. 113 Endogenous stimuli are incited by AMI and act therapeutically to enhance local homing signals. Kollet et al first demonstrated that HSCs used SDF-1 for homing to damaged tissue. 90 They observed that the level of HSC engraftment into liver was greatly enhanced following injury or viral inflammation by elevated MMP-9 activity, which in turn led to increased CXCR4 expression and SDF-1 mediated recruitment of hematopoietic progenitors to the liver. 90 It is well documented that stress signals such as tissue injury or inflammation cause upregulation of SDF-1 in endothelial cells, which promotes the recruitment of stem cells, as demonstrated for heart, 32,41 kidney, 41 and brain. 114 Systemic mobilization and homing to sites of cardiac injury was suggested to be confounded by the trapping of cells in organs such as the spleen 7 ; however, a number of studies have since demonstrated that cytokine therapy can overcome the complications of trapping and demonstrate significant cardiac regeneration in nonsplenectomized animals. 94,115 Malek et al addressed the issue of whether cardiac inflammation plays an important role in successful homing of ES cells to the heart after intravenous delivery in a murine myocarditis model. 116 Maximal engraftment of ES cells occurred at a time of peak inflammatory cytokine production, most notably IL-6, supporting the notion that factors released from the myocardium during an inflammatory response, as occurs in MI, are important for enhancing the homing, migration, and implantation of systemically infused stem cells. Extrapolating to humans, these conclusions raise the possibility that cell transplantation may be a less effective therapy for patients who do not manifest a significant degree of cardiac inflammation, notably patients with end-stage cardiomyopathy. A microarray study aimed at identifying mediators of migration and engraftment of BM-MSCs within the ischemic myocardium 117 revealed 9 complementary adhesion molecules and cytokine receptors, including integrins 1 and 4 and CXCR4. Blockade of integrin 1, but not integrin 4 or CXCR4, significantly reduced BM-MSC number in the infarcted myocardium suggesting that BM-MSCs use a different pathway from HSCs or EPCs for intramyocardial trafficking and engraftment. Cell migration is an essential component of successful mobilization and homing. The actin cytoskeleton is the principal mechanical component responsible for cell guidance and motility. Migration, initiated by a multitude of extracellular stimuli such as extracellular matrix, signaling via chemokine receptors and receptor tyrosine kinases, 118 ultimately requires activation of actin associated proteins, reorganization of the actin cytoskeleton and activation of focal adhesion kinases. Downstream of CXCR4, activation of PI3-kinase, phospholipase C- / protein kinase C cascade and MAPK p42/44 (ERK-1/2), 119 lead to changes in gene expression via the JAK/STAT 120 and nuclear factor B pathways. 119 The importance of JAK signaling in EPC migration for neovascularization was recently documented. 121 EPCs and BM- MNCs from patients with coronary artery disease display significantly diminished migratory responses to SDF-1 and VEGF. Despite showing dysfunctional properties relating to the CXCR receptor, cell surface expression of CXCR4 was comparable to that in healthy donors. However, JAK-2 phosphorylation in response to SDF-1 was significantly reduced in EPCs from patients with coronary artery disease and appears to provide the critical link between SDF/CXCR4 and the angiogenic processes of invasion and migration. Stem Cell Migration Within the Myocardium Cardiac progenitor cells, whether resident or transplanted, migrate through the interstitium of the heart, although the extent of migration and the mechanisms involved are poorly understood. Bone marrow derived HSCs, introduced into remote myocardium 26 and cardiosphere-derived cells injected into the border zone 48 migrated to the infarct region, illustrated by tracking of EGFP and lacz expressing cells, respectively. Although the mechanism of migration was not investigated in these studies, their directional migration toward the scar likely results from secretion of factors by dead or hypoxic cells in a similar manner to stem cell homing, described above. Recent advances in noninvasive imaging technologies have enabled the tracking of progenitor cells, labeled with F-18 fluorodeoxyglucose or iron particles, during homing from the peripheral circulation to ischemic tissues 122 or their migration within the heart following cardiac delivery. 123 As cell therapy is further developed for heart repair, the ability to track cells will prove essential for assessing their capacity to home and migrate to the injury zone. Migration of Endogenous Cardiac Progenitor Cells Several populations of cardiac progenitors residing within the adult heart have now been characterized. However, to maintain stemness, progenitors are required to be retained within a supportive stem cell niche. 124 One of the limitations for cardiac regeneration is that the small progenitor populations within the heart reside in a quiescent state and require reactivation before they can promote regeneration. Indeed some of the factors associated with reactivation and restoration of pluripotency, such as T 4, become upregulated following MI, 52 but even the induced levels are insufficient

Smart and Riley Stem Cell Migration 1163 for regeneration and require exogenous application or therapeutic augmentation of endogenous induction to promote sufficient repair. 125 A rapidly evolving paradigm, and one that holds much promise for therapeutic myocardial regeneration, is the identification of paracrine factors that stimulate endogenous cardiac stem cells to migrate to the site of injury within the heart and differentiate into the cardiomyocyte and vascular cells required to induce neovascularization and repair. Only a few such factors have hitherto been identified, including high-mobility group box protein (HMGB)1 and T 4. It is perhaps significant that the common feature shared by these proteins is the ability to promote cell migration and suggests that migration of progenitors away from their restrictive niche is sufficient to reactivate their proliferation and differentiation. HMGB1 Stimulates Migration of Resident c-kit Cardiac Progenitor Cells HMGB1, a chromatin binding protein is passively released by necrotic cells and, as well as stimulating secretion of proinflammatory cytokines, it may act as a danger signal. This signal represents a chemoattractant for mesangioblasts, vascular smooth muscle cells and fibroblasts, 126 stimulating their migration and homing, via a mechanism that involves cytoskeletal rearrangement. HMGB1 localizes to the leading edges of transformed cells and is secreted into the extracellular space, implying a key role in migration and metastasis. 127 The exact mechanisms that mediate cell attraction by HMGB1 are only gradually being elucidated but its known receptors include the receptor for advanced glycation end products (RAGE) as well as Toll-like receptors 2 and 4, 128 whose downstream signaling pathways include Ras, nuclear factor B, ERK1/2 MAPK, p38 MAPK, SAPK/JNK, and the small GTPases Rac and cdc42. 129 Limana and colleagues explored the potential for HMGB1 to activate a resident population of c-kit cardiac progenitors for postinfarction myocardial regeneration. 130 Administration of HMGB1 into the periinfarct region of the left ventricle resulted in the proliferation and differentiation of endogenous CPCs and the formation of new, although smaller and less mature, cardiomyocytes, within the infarcted region of the LV wall. HMGB-1 induced cardiac regeneration and associated improvement in cardiac performance via effects on resident CPCs with no significant contribution of c-kit cells from the systemic circulation. 130 Although the authors demonstrated significant HMGB1-induced migration of c-kit CPCs, they were unable to detect migration of cardiac progenitors, retrovirally labeled with EGFP, by two-photon microscopy of excised hearts and suggested that technical limitations may have hampered the detection of migrating cells. 130 It remains plausible, therefore, that HMGB1 achieves its regenerative effect, in part, by promoting cardiac progenitor cell migration. Thymosin 4 Stimulates Migration of Resident Epicardium Derived Cells Another source of adult cardiac progenitor cells is the epicardium. 52,55,131 The epicardium is derived by outgrowth of cells from the proepicardial organ, a transient embryonic structure, to overlie the developing heart tube. Following EMT, EPDCs delaminate from the epicardium and subepicardium and migrate into the myocardium and differentiate into cardiac fibroblasts, endothelial, and smooth muscle cells. 132 The identification of T 4 as a factor capable of stimulating adult EPDCs arose from a working hypothesis that molecules that regulate collateral growth in the ischemic heart undoubtedly orchestrate coronary vasculature formation during embryogenesis. T 4 is an actin-monomer sequestering protein required for cell migration via cytoskeletal reorganization and lamellipodia formation. By selectively knocking down T 4in the developing heart, Smart and coworkers recently characterized an essential role for 4 in regulating all 3 key stages of cardiac vessel development. 52 The ability of T 4 to stimulate the developing epicardium in vivo was supported by studies on epicardial explant cultures 52 and examining the differentiated cell types formed following outgrowth. Addition of T 4 significantly increased the numbers of smooth muscle (SM A ) and endothelial (Tie2 ) cells and these cell populations were enhanced further still with the addition of VEGF and FGF7. 52 From these studies, it was concluded that T 4 signals in a paracrine manner from the myocardium to cells of the epicardium. Cells delaminate from the epicardium, undergo EMT, migrate into the myocardium, and differentiate into endothelial cells to form coronary vessels and smooth muscle cells to stabilize the vasculature. Translation of a vascular development role for T 4 to that of angiogenic therapy for coronary artery disease in the adult heart relies on the release of the adult epicardium from a quiescent state and restoration of pluripotency. To investigate the potential for T 4 in this context, the ability of T 4 to induce outgrowth and differentiation of isolated epicardial explants from adult hearts was assessed. It was previously perceived that adult epicardium resides in a state of dormancy, having lost all potential for migration, differentiation and signaling during late embryonic stages, 133 and, indeed, untreated adult explants displayed virtually no detectable outgrowth. In contrast, treatment with T 4 stimulated extensive outgrowth of epicardial cells which, following migration, differentiated into fibroblasts, smooth muscle cells, and endothelial cells, definitive progenitors of the coronary vasculature. These data demonstrate that, under the control of T 4, persistent vasculogenic potential of the adult epicardium may be harnessed for use in therapeutic angiogenesis. In support of these observation, Van Tuyn et al reported that epicardial cells from human adult heart can undergo EMT and obtain characteristics of smooth muscle cells in vitro. 131 A recent study 55 provides evidence that the adult human and mouse epicardium can support a rare population of c-kit stem cells, identical in cell surface marker profile to those previously described in the myocardium. 44 Thus, the adult epicardium may act as a stem cell niche to support not only this rare c-kit but also the more prevalent EPDC lineage and provide EPDCs with potential to contribute to cardiac homeostasis and repair. In considering the epicardium as a source of vascular progenitors, valuable insight may be derived from studies in the zebrafish. Following ventricular resection of the adult fish

1164 Circulation Research May 23, 2008 heart, the epicardium exhibits a rapid and robust response to injury, which includes proliferation and expression of embryonic epicardial markers (Tbx18 and RALDH2). 134 The activated epicardium envelopes the cardiac chambers, including the injured apex and a subpopulation of cells invades the subepicardial space and myocardium to contribute endothelial and smooth muscle cells to form new coronary vessels; this is highly reminiscent of the processes involving the epicardium during mouse embryonic heart development, 135 for which T 4 was required. 52 It is significant therefore that, in a related study, the fish ortholog of T 4 was found to be upregulated in regenerating zebrafish hearts. 136 Taken together, these data, along with the ability of T 4 to mobilize murine adult EPDCs, provide strong support for the potential of T 4 to induce neovascularization and possibly other aspects of myocardial regeneration, for homeostasis 137 and repair following infarct in the adult heart. Cell-Based Therapies: Modes of Delivery Systemic delivery of stem cells as an effective therapy for the injured heart is dependent on successful homing and retention of cells before the secretion of paracrine factors and/or transdifferentiation. There are 2 ways in which cardiac progenitors can be delivered to the heart: either by an intracoronary arterial route or by injection into the ventricular wall via percutaneous endocardial, percutaneous transcoronary venous, or surgical epicardial approaches. Intracoronary delivery enables the application of a maximum dose of cells homogeneously to the site of injury although this mode is less efficient for delivery to nonperfused regions of the infarct related artery. Homing of intraarterially applied progenitor cells requires their extravasation and migration to the surrounding ischemic tissue. Although BMSCs and hematopoietic stem cells can extravasate, 7 this has not been shown for all cell types and larger, less motile cells, such as skeletal myoblasts may even obstruct the microcirculation, leading to embolic myocardial infarction. 8 Direct injection is the preferred delivery method for chronic heart failure patients with considerable scar tissue. Cell homing signals such as SDF-1 and VEGF are expressed at low levels at late stages of disease, limiting any homing potential following intracoronary application. 138 Injection into the injured myocardium is particularly suited for large cells such as myoblasts and mesenchymal stem cells and is not limited by cell uptake from the circulation or embolic risk. However, injection of progenitor cells into necrotic tissue, which lacks both blood flow to provide oxygen and nutrients and healthy surrounding cardiomyocytes to provide paracrine support, reduces graft survival and differentiation. The optimal delivery route for autologous cell transplantation not only varies according to the administered cell type but will be influenced in the future by our ability to enhance the migratory capacity of stem cells. Conclusions How does our knowledge of stem cell migration assist in developing therapies for disease? Previous therapies have attempted to harvest stem cells from the bone marrow, isolate the relevant subpopulation, and readminister them via intravenous injection. This approach seldom delivers the desired clinical benefits. Central to the processes of mobilization of cells from the bone marrow and engraftment into the target organ is the regulation of cell migration. An understanding of the mechanisms involved in initiation and regulation, along with identification of factors that direct these processes, may offer approaches to enhance present strategies involving stem cell engraftment. Moreover, approaches requiring the introduction of exogenous stem cells are hampered by inefficient homing and immune rejection. In the absence of an effective intervention to optimize migration of transplanted cells, a preferable therapeutic strategy might be to stimulate one or more of the identified populations of endogenous cardiac stem cells to initiate repair in situ. Before any proliferation or differentiation, they key initial event required to unleash the potential of these cells is their migration from the stem cell niche to the site of injury. The search is underway, therefore, to identify factors that can revive the potential of these cells to achieve efficient cardiac regeneration. Sources of Funding Our work is supported by the British Heart Foundation and the Medical Research Council. None. Disclosures References 1. 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The Stem Cell Movement Nicola Smart and Paul R. Riley Circ Res. 2008;102:1155-1168 doi: 10.1161/CIRCRESAHA.108.175158 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright 2008 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/102/10/1155 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/