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REVIEW ARTICLE Regenerative therapy for cardiovascular disease Q15

OTMAR PFISTER, GIACOMO DELLA VERDE, RONGLIH LIAO, and GABRIELA M. KUSTER BASEL, SWITZERLAND; AND BOSTON, MASS

Recent insights into myocardial biology uncovered a hereto unknown regenerative capacity of the adult heart. The discovery of dividing cardiomyocytes and the identification and characterization of cardiac stem and progenitor cells with myogenic and angiogenic potential have generated new hopes that cardiac regeneration and repair might become a therapeutic option. During the past decade, multiple candidate cells have been proposed for cardiac regeneration, and their mechanisms of action in the myocardium have been explored. Initial clinical trials have focused on the use of bone marrow cells to promote myocardial regeneration in ischemic heart disease and have yielded very mixed results, with no clear signs of clinically meaningful functional improvement. Although the efficiency of bona fide cardiomyocyte generation is generally low, stem cells delivered into the myocardium act mainly via paracrine mechanisms. More recent studies taking advantage of cardiac committed cells (eg, resident cardiac progenitor cells or primed cardiogenic mesenchymal stem cells) showed promising results in first clinical pilot trials. Also, transplantation of cardiomyogenic cells generated by induced pluripotent stem cells and genetic reprogramming of dividing nonmyocytes into cardiomyocytes may constitute attractive new regenerative approaches in cardiovascular medicine in the future. We discuss advantages and limitations of specific cell types proposed for cell-based therapy in cardiology and give an overview of the first clinical trials using this novel therapeutic approach in patients with cardiovascular disease. (Translational Research 2013;-:1–13) Abbreviations: BMC ¼ bone marrow-derived cell; BMSC ¼ bone marrow stem cell; CADUCEUS ¼ Cardiosphere-Derived Autologous Stem Cells to Reverse Ventricular Dysfunction; CD ¼ Cluster of differentiation; c-kit ¼ receptor for SCF; CSC ¼ cardiac stem cell; ESC ¼ embryonic stem cells; HF ¼ heart failure; iPS ¼ induced pluripotent stem cell; MSC ¼ mesenchymal stem cell; SCF ¼ stem cell factor; SCIPIO ¼ Stem Cell Infusion in Patients With Ischemic Cardiomyopathy; SP ¼ side population

espite significant advances in the treatment of acute myocardial infarction, stroke, and hypertension, cardiovascular disease continues to remain the leading cause of death in developed countries.1

D

As the common final sequel of virtually all cardiac diseases, heart failure (HF) is the major contributor to cardiovascular morbidity and mortality. In the Western world, an estimated 1%–2% of the population experience HF,

From the Department of Biomedicine, University Hospital Basel and University of Basel, Basel, Switzerland; Division of Cardiology, University Hospital Basel, Basel, Switzerland; Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.

Reprint requests: Otmar Pfister, MD, Division of Cardiology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland; e-mail: [email protected].

Conflicts of Interest: All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare.

1931-5244/$ - see front matter Ó 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2013.12.005

Submitted for publication September 17, 2013; revision submitted November 4, 2013; accepted for publication December 5, 2013.

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Extracardiac sources Pluripotent stem cells Pluripo

Blood - EPCs

ESCs*

- Mesangioblasts*

iPSs*

Bone marrow - MSCs Cardiac sources C

- HSCs - EPCs

H Heart - c-Kit+ cells

- SP cells* - BMMNCs

- Sca-1+ cells* - SP cells*

Adipose tissue - MSCs - SP cells* - EPCs*

- Isl-1+* Skeletal muscle - Myoblasts

- Cardiospheres - EDPCs*

- Pericytes*

Fig 1. Sources of cells for cardiac repair. Overview of the various cellular sources considered for cell therapy. BMMNCs, bone marrow mononuclear cells; c-kit, stem cell factor receptor; EDPCs, epicardial-derived progenitor cells; EPCs, endothelial progenitor cells; ESCs, embryonic stem cells; HSCs, hematopoietic stem cells; iPSs, induced pluripotent stem cells; Isl-1, islet 1; MSCs, mesenchymal stem cells; Sca-1, stem cell antigen 1; SP, side population. *Not used in clinical trials.

with the prevalence exceeding 10% in people older than 70 years of age.2 Today, HF has become the leading cause of hospitalization of patients older than 65 years, thus further challenging both public and private healthcare systems across the world. Although today’s medical and device therapies aim to optimize cardiovascular hemodynamics and myocardial remodeling, they do not account for the loss of functional cardiomyocytes, which are at the core of the failing myocardium. In contrast to other muscle tissue, such as smooth muscle and skeletal muscle, the regenerative capacity of the adult myocardium is limited, and thus not sufficient to compensate for cell loss in acute and chronic cardiac injury. Therefore, for decades, the adult heart was considered a postmitotic organ, with virtually no inherent regenerative capacity. With the discovery of dividing cardiomyocytes after myocardial infarction and the documentation of cell chimerism in transplanted hearts, however, this static view of the myocardium was put into question.3,4 Given the prospect of true myocardial regeneration, the quest for potential stem/progenitor cell sources with myogenic and angiogenic potential has generated great enthusiasm in the cardiovascular field. During the past decade, multiple candidate cells have been proposed for cardiac regeneration (Fig 1), and the first clinical trials using cell-based therapies have been performed. We discuss advantages and limitations of specific cell types proposed for cell-based therapy and give an overview of the first clinical trials using this novel therapeutic approach in patients with cardiovascular disease.

THE BONE MARROW AS A POTENTIAL SOURCE OF CARDIOGENIC CELLS

There is a vast body of evidence demonstrating that ischemic cardiac injury stimulates the recruitment and myocardial homing of endogenous bone marrow-derived cells (BMCs).5-9 The inflammatory process associated Q1 with myocardial ischemia induces several conventionally ‘‘mobilizing cytokines,’’ including granulocyte-colony stimulating factor, stem cell factor (SCF), vascular endothelial growth factor, stromal cell-derived factor 1, and erythropoietin.10 Inability to mobilize and home BMCs properly to the heart after myocardial infarction resulted in early cardiac failure and death, as shown in a proofof-concept study using transgenic mice overexpressing mutant receptor for SCF (c-kit).7 Notably, restoring the Q2 capacity for mobilization and homing by bone marrow transplantation with wild-type BMCs could rescue cardiac function in this model. Also, stromal cell-derived factor 1 and its receptor CXCR4 are important for the mobiliza- Q3 tion of BMCs on myocardial infarction.11 Taken together, these translational studies demonstrate clearly an important functional interaction between the bone marrow and the heart in the setting of cardiac injury. The fact that allogeneic and autologous BMCs have the potential, functionally, to replace a radio- or chemotherapy-ablated hematopoietic system has revolutionized the treatment of malignant hemato-oncologic diseases and established bone marrow stem cells (BMSCs) as the epitome of adult stem cells. Early reports that BMSCs may transdifferentiate into nonhematologic

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Cell-mediated cardiac effects Direct effects

Paracrine effects

Attenuation of apoptosis

Recruitment/Activation of resident stem cells

Modulation of matrix remodeling

Scar stabilization

Angiogenesis vasculogenesis

Increase of capillary density

REVERSE REMODELING

Transdifferentiation into cardiomyocytes, endothelial cells and smooth muscle cells

Providing a nichelike environment

Cardiomyogenic rebuilding

IMPROVED CONTRACTILE PERFORMANCE

Fig 2. Mechanisms of action of cell therapy. Postulated mechanisms underlying the beneficial effects of cell therapy on myocardial remodeling and function include indirect (ie, paracrine effects) as well as direct effects of supplied cells.

cells such as skeletal muscle, hepatocytes, neurons, endothelial cells, and even cardiomyocytes prompted attempts to use BMSCs to promote cardiovascular regeneration after myocardial injury.12-14 Preclinical animal studies isolated BMSCs expressing the stem cell receptor c-kit for direct injection into the border zone of infarcted myocardium in small- and largeanimal models.15,16 In these animal models, cell-based therapy using BMSCs showed impressive regeneration of lost myocardium, new formation of capillaries, and improvement of cardiac function. Although later reports refuted true transdifferentiation of BMSCs in cardiomyocytes and claimed cellular fusion as the underlying mechanism for the acquisition of cardiomyogenic phenotypes of BMSCs,17 most animal studies demonstrated consistently preservation of myocardial tissue and improvement of cardiac function after BMSC transplantation.15,18 The cognition that BMSCs improve cardiac function after myocardial infarction without meaningful transdifferentiation into new cardiomyocytes led to the paracrine theory of cardiac protection and regeneration. BMSCs and other adult stem cells have profound paracrine activity that includes secretion of cardioprotective cytokines that inhibit apoptosis, angiogenic factors (eg, vascular endothelial growth factor, basic fibroblast growth factor) that promote neovascularization, and factors that activate resident cardiac stem cells (CSCs)/progenitor cells (insulinlike growth factor).19 Therefore, the regenerative potential of BMSCs may be explained by a combination of the following mechanisms: (1) increase of residual viable cardiomyocytes in the infarct border zone resulting

from antiapoptotic effects of BMSCs,19 (2) induction of angiogenesis and vasculogenesis through stimulation by angiogenic factors secreted by BMSCs and new capillary formation by endothelial progenitor cells,20,21 (3) stimulation of resident CSCs,22 and (4) induction of cardiomyocyte mitosis.23 Potential mechanisms of cardioprotection and regeneration provided by BMSCs are summarized in Fig 2. BMSC-BASED THERAPY IN CARDIOLOGY: TRANSITION FROM BENCH TO BEDSIDE

After promising preclinical animal studies, the transition of BMSC-based therapy from research laboratories to the clinical arena unfolded at a rapid pace. The use of BMSCs has some important advantages in the clinical setting. Bone marrow can be accessed easily, is renewable, and contains a cell mixture of autologous cells with regenerative capacity. Most clinical studies used the mononuclear cell fraction for cardiac transplantation because it contains the full array of BMSC precursor/progenitor cells—namely, hematopoietic stem cells, mesenchymal stem cells (MSCs), endothelial progenitor cells, and side population (SP) cells, all of which were shown to improve cardiac function if transplanted into infarcted myocardium in various animal studies.5,15,24-26 In the clinical setting there are 3 major application routes how cells are transplanted into the heart: (1) direct transepicardial injection during open heart surgery (eg, coronary artery bypass graft surgery),27 (2) transendocardial injection,28 or (3) direct intracoronary injection through an inflated over-the-wire balloon catheter.29 To date, most clinical BMSC-based stem cell trials were

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Abbreviations: BMCs, bone marrow cells; CMR, cardiac magnetic resonance tomography; CSCs, cardiac stem cells; ECHO, echocardiography; ESCs, embryonic stem cells; iPSs, induced pluripotent stem cells; LVEF, left ventricular ejection fraction; MSCs, mesenchymal stem cells; ND, not determined; NS, non significant; SPECT, single photon emission computed tomography. *XXXX. Q12

Moelker et al43 De Silva et al44 Schuleri et al45 Williams et al46 Dawn et al47 Bolli et al48 Johnston et al49 Lee et al50 Timothy et al51 Mauritz et al52 Li et al53 Rajasingh et al54 3 NS 3.8 21 29 ND 5 ND ND 25 4 10–20 NS NS 20 20 10 9 NS 6 13 5–10 5.5 10–15* CMR CMR CMR CMR ECHO ECHO CMR ECHO ECHO CMR SPECT ECHO Intracoronary Intracoronary Intramyocardial Intramyocardial Intracoronary Intracoronary Intracoronary Iintramyocardial Intramyocardial Intramyocardial Intramyocardial Intramyocardial 20/10 7/7 9/6 5/5 24/17 11/10 7/7 18/10 6/6 25/13 9/9 40/20 Pig Pig Pig Pig Rat Pig Pig Pig Mice Mice Pig Mice BMCs BMCs MSCs MSC-CSC mix CSCs CSCs CSCs (cardiospheres) CSCs (cardiospheres) iPSs iPSs iPSs ESCs

Occlusion and reperfusion Occlusion and reperfusion Occlusion and reperfusion Occlusion and reperfusion Occlusion and reperfusion Occlusion and reperfusion Occlusion and reperfusion Occlusion and reperfusion Permanent occlusion Permanent occlusion Occlusion and reperfusion Permanent occlusion

Animal Cell type

Infarct model

Treated/control, n

Delivery route

Imaging method

Increase in LVEF vs control, %

Decrease in scar size vs control, %

References

Q11

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Table I. Summary of proof-of-concept preclinical studies

4

performed in the setting of acute myocardial infarction with direct intracoronary injection as the main cell application route.30 After promising results from initial noncontrolled clinical trials,29,31,32 larger randomized and controlled trials were initiated. However, these well-designed, in some cases even blinded, randomized clinical trials generated very mixed results, with most trials being neutral.33-39 The contradictory outcomes of randomized trials were explained by large variations in cell preparation,40,41 the use of different cell types and cell numbers for transplantation, and the different time points of cell injection.42 To address mechanistic questions in cardiovascular cell therapy, the U.S. National Heart, Lung, and Blood Institute established the Cardiovascular Cell Therapy Research Network. Within this network, the TIME study program was initiated to Q4 address specifically the question of timing of cell therapy. In the TIME study, patients were randomized to receive either intracoronary BMSC therapy or placebo at day 3 or day 7 after acute myocardial infarction. In the LateTIME study, patients were randomized to in- Q5 tracoronary BMSC transplantation or placebo as late as 2–3 weeks postinfarct. Both trials failed to show any recovery of global or regional left ventricular function compared with placebo, regardless of the time point of cell therapy.43,44 Major pre-clinical and clinical trials using BMSC for myocardial regeneration are summarized in Tables I43-54 and II,30,34-39,55-61 respectively. Q6 Today, after more than 10 years of clinical experience and more than 30 randomized controlled trials including more than 2000 patients, the use of BMSCs for cardiac regeneration remains highly controversial. Although this therapy has proved safe, its clinical efficacy seems rather limited, as suggested by the latest meta-analyses.30 There is no reduction of mortality, reinfarction or rehospitalization. At best, there may be a risk reduction for future revascularization and a modest improvement of left ventricular ejection fraction in the range of 3%.30 Therefore, although easily accessible, BMSCs exhibit some inherent biologic limitations that challenge their usefulness for successful regenerative therapy of the myocardium. Among these limitations are (1) poor cell retention and survival in the myocardium regardless of the route of delivery,62,63 (2) the lack of meaningful cardiomyogenic differentiation,17 and (3) the loss of paracrine function associated with age and cardiovascular risk factors.64 As a consequence, new approaches to improve cell survival and preserve paracrine function are currently being explored.65,66 MESENCHYMAL STEM CELLS

In the bone marrow, MSCs are essential components of the microenvironment necessary for the proliferation and differentiation of hematopoietic stem cells. They

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Q13

Table II. Summary of major cell-based clinical trials Trial

Cell type

Bone marrow mononuclear cells BOOST BMCs Jannsens BMCs ASTAMI BMCs REPAIR-AMI BMCs BONAMI BMCs HEBE BMCs FOCUS-CCTRN BMCs TIME BMCs Late TIME BMCs Meta-analysis BMCs MSCs and resident CSCs C-CURE Cardiopoietic MSCs POSEIDON MSCs SCIPIO CSCs CADUCEUS CSCs

Delivery route

Intracoronary Intracoronary Intracoronary Intracoronary Intracoronary Intracoronary Endomyocardial Intracoronary Intracoronary Intracoronary Endomyocardial Endomyocardial Intracoronary Intracoronary

Treated/control, n

30/30 33/34 47/50 101/103 52/49 135/65 61/31 78/41 58/29 1830 32/15 31/no control 16/7 17/8

Days after MI

Imaging method

Change in LVEF vs control, %

Decrease in scar size vs control, %

References

Meyer et al55 Janssens et al36 Lunde et al35 Schachinger et al34 Roncalli et al37 Hirsch et al38 Perin et al39 Traverse et al56 Traverse et al57 Zimmet et al30

6 1 6 3–7 9 3–8 ICMP 3–7 14–21

CMR CMR SPECT, CMR LVA RNA, CMR CMR SPECT, ECHO CMR CMR

NS NS NS 2.5 NS NS NS NS NS 2.7

ND 28 NS ND NS NS NS NS NS ND

ICMP ICMP ICMP 45–90

ECHO CCT ECHO, CMR CMR

7 NS 8 NS

ND 33 24–30 9

Bartunek et al58 Hare et al59 Bolli et al60 Malliaras et al61

Abbreviations: BMCs, bone marrow cells; CCT, contrast-enhanced computed tomography; CSCs, cardiac stem cells; CMR, cardiac magnetic resonance tomography; ECHO, echocardiography; ICMP, ischemic cardiomyopathy; LVA, left ventricular angiogram; LVEF, left ventricular ejection fraction; MI, myocardial infarction; MSCs, mesenchymal stem cells; ND, not determined; NS, not significant; RNA, radionuclide angiogram; SPECT, single photon emission computed tomography.

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represent approximately 0.001%–0.01% of bone marrow nucleated cells.67 MSCs are self-renewing and exhibit multilineage differentiation potential, including the potential to differentiate into cardiomyogenic cells if stimulated properly in vitro.67-69 Some, but not all, reports suggest cardiac engraftment, and the cardiomyogenic and angiogenic differentiation capacity of MSCs in large-animal models of acute and chronic ischemic cardiomyopathy.70-74 In these preclinical models, cellbased therapy with autologous or allogeneic MSCs improved left ventricular remodeling and ejection fraction consistently, and reduced scar size.45,75,76 More important, the beneficial effects observed in preclinical trials were achieved despite the lack of long-term MSC engraftment and limited transdifferentiation capacity of engrafted cells.77,78 Thus, similar to BMSCs, the unique paracrine secretome of MSCs that includes cytokines and growth factors, which inhibit fibrosis and apoptosis, and stimulate angiogenesis and differentiation of tissue-specific stem cells, is probably more important for the observed beneficial effects on cardiac remodeling and function than transdifferentiation of MSCs into functional cardiac cells.79-81 To provide a standardized definition of the human MSC phenotype, the International Society for Cellular Therapy proposed the following criteria to identify and select human MSCs: (1) adherence to plastic culture dishes; (2) expression of the following cell surface markers: cluster of differentiation (CD) 73, CD90, CD105 in the absence of CD34, CD45, HLA-DR, CD14 or CD11 b, CD79a, or CD19 as assessed by fluorescence-activated cell sorter analysis; and (3) in vitro capacity to differentiate into mesodermderived tissues including chondroblasts, osteoblasts, and adipocytes.82 Because of their unique immunophenotype, human MSCs are hardly immunogenic and are thus suited for allogeneic transplantation.83 Clinical phase I/II trials have already been performed to investigate the safety and efficacy of intravenous, intracoronary, or endomyocardial injection of autologous or allogeneic MSCs in acute myocardial infarction and ischemic cardiomyopathy.84-86 In these early clinical trials, the safety of MSC transplantation was documented, and reverse remodeling and improved regional contractility of the infarcted area were observed.86 Because the cardiomyogenic differentiation potential of bone marrow-derived MSCs is limited, recent approaches have aimed at directing MSCs toward cardiopoietic transdifferentiation by exposing them to a cardiogenic cocktail regimen that triggers expression and nuclear translocation of cardiac transcription factors.87 In the multicenter, randomized Cardiopoietic Stem Cell Therapy in Heart Failure (or C-CURE) trial, this novel cell therapy approach was tested in patients

with HF of ischemic origin.58 In the cell therapy arm, primed cardiopoietic MCSs were delivered by endomyocardial injection guided by NOGA electromechan- Q7 ical mapping into areas of dysfunctional but viable myocardium. After 6 months, patients randomized to cell therapy exhibited significant improvement of cardiac function and reduction of adverse ventricular remodeling compared with patients receiving standard care.58 Moreover, cardiopoietic stem cell therapy was associated with better exercise tolerance and quality of life.58 These promising results support the concept of lineage specification in stem cell therapy and provide the basis for larger, randomized, controlled clinical trials testing this novel therapeutic approach. The safety and feasibility of allogeneic MSC transplantation was demonstrated recently in the POSEIDON trial. In this trial, 30 patients with ischemic cardiomyopathy were randomized either to autologous or allogeneic transendocardial injection of allogeneic MSCs.88 MSC injection affected patient functional capacity, quality of life, and ventricular remodeling favorably. Most important, allogeneic MSC transplantation proved safe and was not associated with adverse immune responses, thus opening the door for larger clinical trials using off-the-shelf allogeneic MSCs. Major preclinical and clinical trials using MSCs for myocardial regeneration are summarized in Tables I and II, respectively. RESIDENT CARDIAC STEM CELLS

In parallel with the attempts to regenerate the heart with exogenous stem cells, the search for potential resident CSCs became another focus of regenerative cardiovascular research. In 2002, Hierlihy et al89 described for the first time the presence of a stem cell-like population in the heart. Based on the specific ability to efflux the DNA binding dye Hoechst, these authors identified socalled SP cells with cardiomyogenic potential in the postnatal heart. A hallmark of SP cells is their high expression of ABC transporters, including Abcg2 (also known as bcrp1) and Abcb1 (also known as multidrug resistance protein 1), which allow for efficient efflux of cytotoxins and dyes.90 SP cells were identified and characterized originally in the bone marrow, where they encompass the majority of long-term repopulating cells within the fraction of hematopoietic stem cells.91 Later, SP cells with organ-specific progenitor cell capacities could be isolated from solid organs, including pancreas, pituitary, testis, mammary gland, lung, liver, skeletal muscle, and, eventually, the heart.92,93 In contrast to bone marrow SP cells, SP cells isolated from the myocardium lack expression of hematopoietic markers, but express cardiac-specific transcription factors such as Nkx 2.5, GATA 4, and Mef2.94 In mice, there is a gradual decrease in cardiac SP cells from neonatal

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(9.4 6 1.2%) to adult age (0.8 6 0.1%).90 Although SP cells can be isolated from human hearts, their exact amount is not known. Murine SP cells can be propagated in large numbers and stored frozen for further use.90 Data from our group demonstrated not only the capacity for biochemical, but also functional cardiomyogenic, differentiation of cardiac SP cells.94 Moreover, we showed that, on myocardial infarction, cardiac SP cells within the infarct border zone are activated and undergo cell division, presumably in an attempt to participate in myocardial regeneration.6 Indeed, if injected intravenously, cardiac SP cells home to the injured myocardium and differentiate into cardiomyocytes, as shown in a myocardial cryoinjury rat model.95 Since the first description by Hierlihy et al,94 various independent groups demonstrated consistently the differentiation of cardiac SP cells into cardiomyocytes and endothelial cells in vitro and in vivo, thereby establishing this unique cell population as a potential source of cardiac progenitor cells.94-98 In addition to cardiac SP cells that are isolated based on functional properties such as the Hoechst efflux phenotype, other cardiac progenitor cell populations are identified according to the expression of stem cell surface markers, including the receptor for SCF (c-kit) and stem cell antigen 1 or their ability to form socalled cardiospheres.99-102 In 2003, Beltrami et al100 characterized thoroughly a population of primitive cardiac cells expressing c-kit. C-kit-expressing cardiac cells were clonogenic, and underwent self-renewal and differentiation into all 3 cardiac cell lineages (cardiomyocytes, endothelial cells, and vascular smooth muscle cells). By demonstrating these ‘‘stemness’’ criteria, the seminal work by Beltrami et al100 suggested for the first time the presence of true CSCs in adult myocardium. Recently, Ellison et al103 demonstrated in different rodent models of diffuse myocardial damage that c-kit and CSCs are necessary and sufficient for the regeneration and repair of myocardial damage. The identification and characterization of resident CSCs changed completely the classic view of the heart as a postmitotic organ. Within the new concept, CSCs maintain cardiac cell homeostasis during normal development and pathophysiological states by replacing dying or dysfunctional cells to maintain global cardiac function. Recent cell tracking studies, using highly sensitive tools such as genetic fingerprinting and 14C dating, confirmed turnover of endogenous cardiomyocytes and replacement of lost cardiomyocytes by newly formed cardiomyocytes in mammals and humans.104,105 In the case of acute myocardial infarction, these resident CSCs/progenitor cells are activated and migrate into the site of injury to become a small part of regenerated myocardium. Because regeneration of an infarcted heart necessitates massive

7

cell replenishment, possibly on the order of a billion cells,106 the endogenous regenerative capacities of resident CSCs are overwhelmed and insufficient for selfrepair. Because of their cardiac source and their potent cardiomyogenic potential, in vitro expanded resident CSCs became a promising new cell type for cellular therapy in acute and chronic ischemic heart disease. In animal models, CSCs injected into the myocardium or the infarct-related artery gave rise to all 3 major cardiac cell types and thereby improved cardiac remodeling and function.47,49,50,107 An inherent disadvantage of autologous resident CSCs is, however, that they require several weeks of ex vivo expansion in culture before sufficient cell numbers are at hand for clinical application. Thus, their use in the acute setting, such as acute myocardial infarction, is limited. Recently, the results of the first clinical pilot trials using autologous CSC transplantation in patients with ischemic cardiomyopathy have been reported. The Stem Cell Infusion in Patients With Ischemic Cardiomyopathy (SCIPIO) trial focused on patients with severely reduced left ventricular ejection fraction resulting from ischemic cardiomyopathy who underwent aortocoronary bypass surgery.60 At the time of surgery, isolation of c-kit-positive CSCs was performed from the right atrial appendage, which is removed routinely during this type of surgery. Isolated c-kit-positive CSCs were then expanded in vitro over several weeks to yield a total cell number of approximately 2 million cells. Three to 4 months after bypass surgery, patients randomized to cell therapy were transplanted with 0.5 million–1 million cells via the intracoronary route. The interim analysis of the first 23 randomized patients of this ongoing study demonstrated significant improvement of symptoms, quality of life, and left ventricular ejection fraction in cell-treated patients at 4 months of followup.60 More important, in this study, cardiac magnetic resonance tomography revealed a significant reduction of myocardial scar after 4 months that was reduced further after 12 months, suggesting evidence of myocardial regeneration within formerly infarcted areas in CSC-treated patients.108 In the Cardiosphere-Derived Autologous Stem Cells to Reverse Ventricular Dysfunction (CADUCEUS) trial, autologous cardiospherederived cells grown from endomyocardial biopsies were used for intracoronary transplantation into the infarct-related artery in patients with postmyocardial infarction ventricular dysfunction.109 Also in this trial, cardiac magnetic resonance tomography showed significantly reduced myocardial scar mass at 6 months and 12 months in cell-treated patients as opposed to control patients, in whom the myocardial scar remained unchanged. However, in contrast to SCIPIO, resident stem cell therapy in CADUCEUS did not improve left

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Isolation method

In vitro differentiation

In vivo differentiation

Species

Developmental stage

Sca-11, CD341, CD312,* CD452

FACS

CMC, EC, SMC

CMC, EC, SMC

Mice, pig, rat, human

Embryonic, neonatal, adult

Not determined

c-kit1

Sca-11, Lin2, CD452, CD312, CD342

FACS, MACS

CMC, EC, SMC

CMC, EC, SMC

Mice, pig, rat, dog, human

Embryonic, neonatal, adult

[LVEF, [REF, YLVNM, [LVVM

Sca-11

CD342, Lin2, CD452, c-kit-, SP

FACS, MACS

CMC, EC

CMC, EC

Mice, dog, human

Neonatal, adult

[LVEF, YLVV

FACS

CMC, EC, SMC

Not determined

Mice, rat, human

Embryonic, neonatal, adult

Not determined

FACS

CMC

CMC, EC

Rat, sheep

Embryonic, adult

[LVEF

Enzymatic digestion, culturing, handling Culturing, handling

CMC, EC, SMC

CMC, EC, SMC

Mice, rat, pig, human

Neonatal, adult

YScar mass, [LVVM

CMC, EC, SMC, FB

CMC (?), EC, SMC, FB

Mice, human

Fetal, adult

Not determined

Surface markers

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Isl-11

SSEA-11 Cardiospheres

EDPCs

Lin2, CD452, c-kit2, CD342, CD312, Sca-12 SSEA1, Oct3/41, c-kit2, Sca-12† c-kit1, Sca-11, CD341, CD311

c-kit1, CD341, CD452

Impact on infarction

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Table III. Overview of reported cardiac stem/progenitor cells References

Hierlihy et al,89 Martin et al,96 Pfister et al,94 Mouquet et al,6 Meissner et al,110 Oyama et al,95 Liang et al98 Beltrami et al,100 Linke et al,111 Bolli et al,60 Chugh AR et al,108 Ellison et al103 Oh et al,99 Matsuura et al,112 Linke et al,111 Wang et al,113 van Vliet et al,102 Liang et al98 Laugwitz et al,114 Moretti et al115 Ott et al,116 Hou et al117 Messina et al,101 Smith et al,118 Johnston et al,48 Makkar et al109 Limana et al,119 Ruiz-Villalba et al120 Translational Research - 2013

Abbreviations: c-kit, stem cell factor receptor; CMC, cardiomyocyte; EC, endothelial cell; EDPCs, epicardial-derived progenitor cells; FACS, fluorescence-activated cell sorting; FB, fibroblast; Isl-1, islet-1; MACS, magnetic-activated cell sorting; LVEF, left ventricular ejection fraction; LVNM, left ventricular nonviable mass; LVV, left ventricular volume; LVVM, left ventricular viable mass; REF, regional ejection fraction; Sca-1, stem cell antigen 1; SMC, smooth vascular muscle cell; SP, side population; SSEA, stage-specific embryonic antigen 1. *Martin et al96 found no expression of CD31, whereas Pfister et al94 reported 2 subpopulations of CD311 and CD312 cells among cardiac SP. These differences may be a result of differences in isolation procedures. † Initially negative for c-kit and Sca-1.

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Table IV. Advantages and disadvantages of specific cell types for cardiovascular regeneration Availability in the clinical setting Cell type

BMC MSC CSC iPS ESC

Safety concerns

Availability

Autologous

Allogeneic

Off the shelf

Efficacy

Need for immunosuppression

Teratoma formation

111 111 1 1 1

1 1 1 1 2

2 1 2 1 1

2 111 2 1 1

2/1 1 1(1) 11 11

2 2 2 2/1 1

2 2 2 11 11

Abbreviations: BMC, bone marrow cell; CSC, cardiac stem cell; ESC, embryonic stem cell; iPS, induced pluripotent stem cell; MSC: mesenchymal stem cell.

ventricular ejection fraction nor HF symptoms or quality of life.109 Both trials, SCIPIO and CADUCEUS, proved that intracoronary CSC transplantation was safe. There were no incidence of malignant arrhythmias or sudden cardiac death and no development of cardiac tumors during follow-up. Despite the impressive reduction in myocardial scar mass in both studies and significant improvement in cardiac function and patient symptoms in SCIPIO, these results have to be interpreted with great caution in light of the extremely limited patient numbers and the lack of blinded study design. Larger randomized trials with a placebo-controlled, double-blinded design are needed to confirm these initial promising results and to pave the way for resident CSC transplantation into clinical practice. An overview of reported CSCs/ progenitor cells is given in Table III.110-120 Major preclinical and clinical trials using CSCs for myocardial regeneration are summarized in Tables I and II, respectively. GENERATION OF CARDIOMYOCYTES BY GUIDED DIFFERENTIATION OF MANIPULATED CELLS

Because the intrinsic regeneration and repair of the adult heart after myocardial infarction is rather limited, attempts to force pluripotent stem cells into cardiomyogenic differentiation before intracardiac transplantation are currently being undertaken. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSs) are among the cell types with profound cardiomyogenic differentiation capacity, if manipulated properly in vitro.121,122 Extensive studies of ESC-derived cardiomyocytes have confirmed proper cardiomyocyte morphology, including organized myofibrils, and expression of gap junction.121,123 ESC-derived cardiomyocytes are, however, less mature than adult cardiomyocytes.124 This immature phenotype may be advantageous for transplantation, because it allows for further proliferation and maturation of transplanted cells within the myocardium. As demonstrated by Laflamme et al,125 human ESCderived cardiomyocytes maintain proliferative capacity and mature structurally over time when transplanted into the rat heart. To date, transplantation studies injecting

murine or human ESC-derived cardiac progenitors consistently showed high myocardial engraftment rate, and significant augmentation of cardiac function and capillary density.126,127 Therefore, human ESC-derived cardiomyocytes could serve theoretically as an allogeneic cell source that can be cryopreserved, stored, and used when needed as an off-the-shelf cellular product. However, the pluripotency of ESCs and their allogeneic origin may also produce undesirable effects. Teratoma and tumor formation have been reported after ESC injection, and immunosuppression is required to prevent graft rejection.128 Because the ethical hurdle for human ESC manipulation is enormous, ESCs will hardly become an acceptable cell source for cardiac regeneration in the clinical setting. The recently reported iPSs exhibit comparable pluripotency with ESCs. In contrast to ESCs, however, iPSs can be generated by cell reprogramming technology from autologous fibroblasts and thus are not afflicted with ethical concerns.129,130 The feasibility and therapeutic efficacy of human iPS-derived cardiomyocyte sheets for regenerative therapy was demonstrated recently in a porcine model of ischemic cardiomyopathy.131 In this study, cell transplantation improved cardiac performance significantly and attenuated left ventricular remodeling. Transplanted human iPS cardiomyocytes were detectable as long as 8 weeks after transplantation, but long-term survival was poor. Alternatively, cardiac fibroblasts can be converted directly into cardiac myocytes using advanced cell reprogramming technology.132 Recently, in vivo reprogramming of endogenous fibroblasts into cardiomyocytes was proposed as a novel approach of regenerative therapy after myocardial infarction.133 Forced myocardial expression of GATA4, HAND2, MEF2 C, and TBX5 by retroviral delivery resulted in reprogramming of dividing nonmyocytes to functional cardiaclike myocytes in a mouse model of myocardial infarction. This in vivo reprogramming strategy improved cardiac function and reduced adverse ventricular remodeling after myocardial infarction. Although the in vivo cardiomyogenic reprogramming efficiency is still low with current technologies,

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this novel approach seems promising because it would allow for cell transplantation-free endogenous cardiac regeneration. As with ESCs, however, iPSs come along with important clinical caveats, such as the potential for tumor and teratoma formation.134 Table I gives an overview of preclinical trials using ESCs and iPSs for myocardial regeneration. Taken together, reprogramming technologies open new and promising avenues for cell-based therapies in cardiovascular medicine. However, further studies in large-animal models are needed, and important practical hurdles have to be overcome (eg, low cardiomyocyte generation efficiency, teratoma formation) before translation of this approach into clinical practice. FUTURE DIRECTION IN REGENERATIVE THERAPY FOR CARDIOVASCULAR DISEASE

From a clinical point of view, an ideal cell candidate for cardiovascular regeneration should exhibit the following characteristics: (1) easy access to cell source and timely availability; (2) profound cardioprotective, cardiomyogenic, and angiogenic potential independent from patient age and cardiovascular risk factors; (3) durability in harsh environments (eg, ischemia, oxidative stress); and (4) excellent safety profile (no potential for teratoma formation, immunoreactivity, or arrhythmias). In the light of these clinical requirements, all the previously mentioned cell types have individual advantages and inherent shortcomings, as summarized in Table IV. Despite the enormous scientific achievements in the field, there is still a great need to understand more completely the cellular and molecular mechanisms responsible for stem cell-based cardiac regeneration and repair. Of highest priority is the identification of the cell type and delivery route best suited for cellular therapy in the clinical setting. Also, because retention of stem cells in the myocardium is low, regardless of cell type and delivery route, new strategies to enhance cell engraftment and differentiation are needed. In this regard cardiac-specific decellularized matrices and biopolymers may serve as attractive vehicles to deliver stem cells to the heart. To enhance cell-based therapeutic responses, combinations of 2 cell types with documented positive interactions might be a novel therapeutic strategy, as shown recently in a porcine model of myocardial infarction for the combination of human CSCs with human MSCs.46 In this preclinical study, the combination of the 2 cell types was more effective at reducing infarct size and restoring cardiac function than either cell type alone. Based on these findings, human clinical trials using combination cell therapy are currently in the planning phase. As an alternative to cell transplantation, transplantation of cell-free matrices delivering cardiopoietic factors via sustained release

may be deployed to enhance intracardiac paracrine effects and to activate the intrinsic regeneration capacity of the heart. This would allow for timely treatment, thus obviating the need for time consuming expansion of candidate cells for transplantation. Ultimately, from the clinician’s point of view, cell-based therapies for cardiovascular regeneration have to prove effective in well-designed, randomized, controlled, and doubleblinded clinical trials before their use in clinical practice can be recommended.

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Regenerative therapy for cardiovascular disease.

Recent insights into myocardial biology uncovered a hereto unknown regenerative capacity of the adult heart. The discovery of dividing cardiomyocytes ...
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