NOVEL THERAPEUTIC STRATEGIES

Biodrugs 2008; 22 (6): 361-374 1173-8804/08/0006-0361/$48.00/0 © 2008 Adis Data Information BV. All rights reserved.

Embryonic Stem Cell Transplantation Promise and Progress in the Treatment of Heart Disease Feixiong Zhang and Kishore B.S. Pasumarthi Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada

Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 1. Embryonic Stem (ES) Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 2. In Vitro Cardiac Differentiation of ES Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 3. Electrophysiologic Properties of ES Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 4. Structural and Functional Heterogeneity of ES-Derived Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 5. Enrichment Methods for ES-Derived Myocardial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 6. ES-Cell Transplantation for Cardiac Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 7. Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

Abstract

Cardiovascular diseases remain the leading cause of death worldwide, and the burden is equally shared between men and women around the globe. Cardiomyocytes that die in response to disease processes or aging are replaced by scar tissue instead of new muscle cells. Although recent reports suggest an intrinsic capacity for the mammalian myocardium to regenerate via endogenous stem/progenitor cells, the magnitude of such a response appears to be minimal and has yet to be realized fully in cardiovascular patients. Despite the advances in pharmacotherapy and new biomedical technologies, the prognosis for patients diagnosed with end-stage heart failure appears to be grave. While heart transplantation is a viable option, this life-saving intervention suffers from an acute shortage of cardiac organ donors. In view of these existing issues, donor cell transplantation is emerging as a promising strategy to regenerate diseased myocardium. Studies from multiple laboratories have shown that transplantation of donor cells (e.g. fetal cardiomyocytes, skeletal myoblasts, smooth muscle cells, and adult stem cells) can improve the function of diseased hearts over a short period of time (1–4 weeks). While long-term follow-up studies are warranted, it is generally perceived that the beneficial effects of transplanted cells are mainly due to increased angiogenesis or favorable scar remodeling in the engrafted myocardium. Although skeletal myoblasts and bone marrow stem cells hold the highest potential for implementation of autologous therapies, initial results from phase I trials are not promising. In contrast, transplantation of fetal cardiomyocytes has been shown to confer protection against the induction of ventricular tachycardia in experimental myocardial injury models. Furthermore, results from multiple laboratories suggest that fetal cardiomyocytes can couple functionally with host myocytes, stimulate formation of new blood vessels, and improve myocardial function. While it is neither practical nor ethical to test the potential of fetal cardiomyocytes in clinical trials, embryonic stem (ES) cells serve as a novel source for generation of unlimited quantities of cardiomyocytes for myocardial repair. The initial success in the application of ES cells to partially repair and improve myocardial function in experimental models of heart disease has been quite promising. However, multiple hurdles need to be crossed before the potential benefits of ES cells can be translated to the clinic. In this review, we summarize the current knowledge of cardiomyocyte derivation and enrichment from ES-cell cultures and provide a brief survey of factors increasing cardiomyogenic induction in both mouse and human ES cultures. Subsequently, we summarize the current state of research using mouse and human ES cells for the treatment of heart disease in various experimental models. Furthermore, we discuss the challenges that need to be overcome prior to the successful clinical utilization of ES-derived cardiomyocytes for the treatment of end-stage heart disease. While we are optimistic that the researchers in this field will sail across the hurdles, we also suggest that

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a more cautious approach to the validation of ES cardiomyocytes in experimental models would certainly prevent future disappointments, as seen with skeletal myoblast studies.

Cardiomyocyte division stops in the mammalian heart during early postnatal life.[1,2] As a result, myocytes that die in response to aging as well as pathologic insults are replaced by scar tissue instead of new muscle cells. Furthermore, overactivation of compensatory signals from the sympathetic and renin-angiotensin systems can compromise myocyte viability and cardiac performance, and ultimately cause heart failure. Cardiovascular diseases (e.g. myocardial infarction (MI) and congestive heart failure) account for the death of more people than any other disease. Although recent reports suggest an intrinsic capacity for the mammalian myocardium to regenerate via endogenous stem/progenitor cells, the magnitude of such a response appears to be minimal and yet to be realized fully in the case of cardiovascular patients. According to the latest data from Statistics Canada, approximately 32% of all male deaths and 34% of all female deaths in 2002 were due to cardiovascular disease.[3] While organ transplantation may extend the life of patients diagnosed with end-stage heart failure, this approach suffers from a severe shortage of donor availability. New biomedical technologies have facilitated the development of a number of life-saving drugs and surgical interventions for patients with end-stage heart failure. However, current drug therapies can increase the life expectancy of heart failure patients by only 2–3 years.[4] Data from a large number of cardiovascular clinical trials indicate that pharmacologic therapies can reduce patient mortality by 25–30%.[5] For patients with acute MI, percutaneous coronary intervention combined with fibrinolytic drugs can reduce in-hospital mortality rates to less than 10%.[6] Similarly, life-saving devices such as defibrillators and left-ventricular assist devices can reduce mortality rates by 20–50%.[7,8] Nonetheless, given the increasing incidence of heart failure and cardiovascular-related deaths around the globe, heart disease patients may benefit from the development of new life-saving interventions. Donor cell transplantation is emerging as a promising strategy to regenerate diseased myocardium.[9] Studies from multiple laboratories have shown that transplantation of donor cells (fetal and embryonic stem [ES] cell-derived cardiomyocytes, skeletal myoblasts, smooth muscle cells. and adult stem cells) can improve the function of diseased hearts.[9] In general, paracrine factors secreted from the transplanted cells are thought to play key roles in stimulating angiogenesis and improving the function of the engrafted myocardium. Among these donor cells, skeletal myoblasts and bone marrow stem cells (BMSC) hold the highest potential for implementation of autologous therapies. Following intra-cardiac skeletal myoblast transplantation, a cohort of patients with severe heart failure showed improvement in the ejection fraction.[10] © 2008 Adis Data Information BV. All rights reserved.

However, this intervention led to an increased arrhythmic risk in several patients.[10] Similarly, the initial positive results from the BMSC clinical trials remain highly controversial.[9,11,12] In contrast, transplantation of fetal cardiomyocytes was shown to confer protection against the induction of ventricular tachycardia in experimental myocardial injury models.[13] Furthermore, results from multiple laboratories suggest that fetal cardiomyocytes can functionally couple with host myocytes, stimulate formation of new blood vessels, and improve myocardial function.[13-18] While it is neither practical nor ethical to test the potential of fetal cardiomyocytes in clinical trials, ES cells may represent a novel source of cardiomyocytes for the treatment of ischemic heart disease. In this review, we summarize the current knowledge of cardiomyocyte derivation from ES-cell cultures and provide a survey of ES-cell transplantation studies in models of experimental heart disease. Furthermore, we discuss potential challenges in the clinical realization of ES-derived cardiomyocytes for the treatment of patients with heart failure. 1. Embryonic Stem (ES) Cells ES cells were first derived in 1981 from the inner cell mass (ICM) of pre-implantation-stage mouse embryos (blastocysts) by two independent groups.[19,20] Subsequently, ES cells were derived from several other species.[21-24] In 1998, Thomson et al.[25] were the first to propagate human ES cells. Mouse ES cells can be maintained in an undifferentiated state by growing them on mitomycin-treated mouse embryonic fibroblasts (MEFs) and/or by supplementing cultures with leukemia inhibitory factor (LIF).[26,27] In contrast, human ES cells are cultured on irradiated MEFs and supplemented with basic fibroblast growth factor.[28] When maintained in an undifferentiated state, these cells can proliferate indefinitely and carry a normal karyotype. ES cells are considered as pluripotent stem cells, which can give rise to cells of all three primary germ layers.[29] In the absence of conditions that maintain them in an undifferentiated state, ES cells will spontaneously differentiate to form multicellular aggregates known as embryoid bodies (EBs). Over a period of time, aggregated cells in EBs can form elements of all three germ layers. Consistent with their in vitro pluripotent nature, injection of mouse ES cells in syngeneic or immunocompromised animals can give rise to tumors containing derivatives of all three germ layers (teratomas).[30] Existing human ES-cell lines may contain chromosomal abnormalities and are likely to form teratomas or other cell lineages after transplantation. Hence, high-fidelity methods for fractionation of differentiated cells from stem cell cultures should form the focus of future ES cell-based therapies. Biodrugs 2008; 22 (6)

ES-Cell Transplantation and Myocardial Repair

Although embryo/ICM-derived ES cells serve as a reliable source for cell-based therapies, clinical use of these stem cells for the treatment of human disease is currently prohibited because of several political, scientific, and ethical reasons (reviewed by Das et al.[31]). These concerns are mainly related to the destruction of human embryos for derivation of ES cells, immune rejection, and safety issues of human cells grown on mouse feeder cell layers (i.e. MEFs). In this regard, major efforts are being pursued to derive ES cells through alternative mechanisms. These alternative approaches include reprogramming of adult somatic cells into pluripotent stem cells using methods such as the somatic cell nuclear transfer technique (SCNT),[32,33] derivation of ES cells from single blastomeres without destruction of whole embryos,[34,35] and genetic conversion of fibroblasts into stem cells.[36-38] While all three approaches are technically challenging, the SCNT technique is yet to be realized for derivation of human ES cells. In contrast to the first two methods, genetic conversion of fibroblasts does not rely on the use of either human oocytes or fertilized embryos and hence is rapidly gaining popularity. It has been shown that the introduction of four genes, POU5F1 (Oct3/4), SOX2, MYC, and KLF4 into adult mouse or human fibroblasts can give rise to pluripotent stem cells with much higher efficiency compared with the efficiencies of SCNT and blastomere approaches.[36-39] The ES cells resulting from genetic conversion of fibroblasts are referred to as induced pluripotent stem (iPS) cells. Similar to the ICM-derived ES cells, iPS cells can give rise to cells of all three germ layers, form teratomas, and also give rise to chimeric animals when injected into blastocysts. Furthermore, the iPS approach eliminates the immune rejection associated with cell-based therapies, as it facilitates generation of patient-specific stem cells from their own somatic cells. However, the presence of MYC or random genomic integration of exogenous genes in differentiated cells derived from iPS cells is believed to increase the risk of cellular transformation.[31] Furthermore, iPS cells have been shown to exhibit some differences in DNA methylation status compared with ICM-derived ES cells.[39] Current efforts are aimed at finding alternative gene combinations for genetic conversion of adult fibroblasts into iPS cells to lower the risk of transformation.[40] Availability of new-generation iPS cells could certainly reduce some of these pending concerns. Additional studies should be conducted to fully characterize electrophysiologic features of iPS-derived cardiomyocytes, optimize methods of cardiomyocyte enrichment, and also test their potential for the regeneration of diseased myocardium similar to the ongoing validation studies performed on ICM-derived ES cells. 2. In Vitro Cardiac Differentiation of ES Cells The cardiomyogenic potential of mouse and human ES-cell lines has been well documented.[29,41-43] For cardiac differentiation of mouse cell lines, dispersed ES cells are grown in suspension © 2008 Adis Data Information BV. All rights reserved.

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culture with no LIF and allowed to form EBs. After 3–4 days, EBs are transferred to plastic culture dishes and allowed to attach. Regions of cardiogenesis can be readily identified by the presence of spontaneous contractile activity 8–10 days after EB attachment.[26,43] The procedure for in vitro cardiac differentiation of human ES cell lines is identical to that of mouse ES cells, but the time schedule for each step is a little longer (i.e. 5–6 days for EB aggregation and 10–14 days for beating).[41] Under normal culture conditions, about 15–20% of mouse EBs can spontaneously differentiate into cardiomyocytes. In contrast, the cardiomyogenic differentiation rates of existing human ES-cell lines are relatively low and account for only 5–20%.[26,41,44] In order to increase cardiomyogenic induction, different factors were used during the differentiation process of both mouse and human ES cells (see tables I and II). These agents include growth factors/hormones (e.g. transforming growth factor-β, vascular endothelial growth factor, insulin growth factor-1, bone morphogenetic protein-2, oxytocin) and chemical reagents (e.g. H2O2, dimethyl sulfoxide, nitric oxide, 5-azacytidine). A number of studies confirmed that the expression profiles of transcription factors, ion channels, and myocyte-specific proteins during ES cardiogenesis are similar to the stage-dependent changes observed during embryonic and neonatal development.[45-47] The ability of ES cells to differentiate into atrial,[46,48,49] ventricular,[46,49-51] and pacemaker cells[52-54] was documented by monitoring marker gene expression via immunostaining or reverse transcription-PCR, as well as action potential (AP) recordings. In addition to the conventional suspension culture of EBs in culture dishes, efforts are underway to facilitate large-scale propagation of ES cardiomyocytes using engineered heart tissue constructs,[55,56] rotary cell culture systems,[57] and bioreactor technologies.[58-60] Although preliminary studies are encouraging, further studies are required, particularly with human ES cells, to optimize conditions and growth factor combinations for maximal yield of cardiomyocytes using these new technologies. 3. Electrophysiologic Properties of ES Cardiomyocytes ES-derived cardiomyocytes can be classified as pacemaker, atrial, ventricular, nodal, His, and Purkinje-like cells based on their AP characteristics. In mouse ES cultures, cells with pacemaker-like AP were found only at the early and intermediate stages, whereas cells with atrial, ventricular, or nodal types of APs were identified primarily in the later stages of differentiation in ES-cell cultures.[46,51] Pacemaker-like cells were identified by a relatively depolarized and short-lasting AP of smaller amplitude.[46,51] Cells with a relatively stable resting potential and a characteristic notch and plateau phase were designated as Purkinje-like cells.[46,51] In contrast, ES-derived atrial cells exhibited a stable resting potential and a short-lasting AP. Ventricular Biodrugs 2008; 22 (6)

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Table I. Factors increasing cardiomyogenesis of mouse embryonic stem (ES) cells ES-cell line (mouse)

Factor

Reference

D3

RA

50

D3

H2O2, menadione

61

CGR 8

BMP2

62

CGR 8

TGFβ

62

D3

VEGF

63

J1

FGFR1

64

ht7

RA

65

D3

PDGF-BB, SPP

66

R1

Avian precardiac endoderm/mesoderm

67

D3

IGF1

68

D3

Nitric oxide

69

ht7

TSA

70

E14

RXR agonist, BMP4, and serum-free condition

71

CGR 8

BMP2

72

CGR 8

TGFβ2

73

D3

RGD oligopeptides (inhibitor of β1 integrin), lithium, exoenzyme C3

74

D3, ATCC CRL 1934

Small signaling molecules (ryanodine, methoxyverapamil, and cyclosporine [ciclosporin] A)

75

D3

Icariin, icaritin, desmethylicartin

76

D3

BMP2 combined with visceral endoderm-like cells

77

D3

cAMP

78

D3

Ascorbic acid

79

D3

PPARα

80

CGR 8, R1

Chibby

81

Royan B1

Oxytocin

82

Royan B1

bFGF

83

Royan B1

BMP4

84

AB2.2

Desmin

85

D3

HGF

86

R1

RA

87

CGR 8

TGFβ1, BMP2, BMP4, activin-A, FGF2, FGF4, IL-6, IGF1, IGF2, VEGF-A, EGF

88

NA

HRG-β1

89

D3

Oxytocin

90

bFGF = basic fibroblast growth factor; BMP = bone morphogenetic protein; cAMP = cyclic adenosine 3′,5′-monophosphate; EGF = epidermal growth factor; FGF = fibroblast growth factor; FGFR = FGF receptor; HGF = hepatocyte growth factor; HRG-β1 = heregulin-β1; IGF = insulin-like growth factor; IL = interleukin; NA = not available; PDGF-BB = platelet-derived growth factor-BB; PPARα = peroxisome proliferator-activated receptor α; RA = retinoic acid; RGD = arginine, glycine, aspartic acid tripeptide; RXR = retinoid X receptor; SPP = sphingosine-1-phosphate; TGF = transforming growth factor; TSA = trichostatin A; VEGF = vascular endothelial growth factor.

cells exhibited a relatively stable, negative resting potential and a [46,51]

long plateau phase.

Similar AP recordings characteristic of

atrial, ventricular, and nodal-like cardiomyocytes were also documented in human ES cultures.[104,105] In addition to the AP recordings, several investigators used a microelectrode array technique to measure changes in field potentials (extracellular recordings) of ES-derived cardiomyocytes in response to pharmacologic treatments as well as during differentiation.[52,105,106] © 2008 Adis Data Information BV. All rights reserved.

4. Structural and Functional Heterogeneity of ES-Derived Cardiomyocytes As cardiomyogenesis in ES cultures is a fairly random process, it is likely that EBs from a given culture stage would contain cardiomyocytes at different stages of development. Although ESderived cardiomyocytes have been shown to express a number of cardiomyocyte-restricted markers and respond to β-adrenergic and muscarinic agonists, it was suggested that the phenotypes of cardiomyocytes in differentiating mouse EBs are reminiscent of Biodrugs 2008; 22 (6)

ES-Cell Transplantation and Myocardial Repair

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cardiomyocytes from mouse hearts at embryonic day 8.75–9 (E8.75–9), where chamber formation has just started.[107,108] This notion is consistent with other studies that documented major differences in Ca2+-induced Ca2+ release (CICR) between human ES-derived cardiomyoctyes and adult ventricular cardiomyocytes.[109,110] It was shown that the [Ca2+]i transients and contraction of human ES-derived cardiomyocytes depended on transsarcolemmal Ca2+ influx but not on the classical CICR mechanism.[109,110] Furthermore, human ES-derived cardiomyocytes did not express regulatory proteins involved in Ca2+ homeostasis such as phospholamban, junctin, triadin, and calsequestrin, which are normally expressed in mature adult cardiomyocytes.[109,111] Collectively, these studies suggest that ES-derived cardiomyocytes harbor an immature sarcoplasmic reticulum (SR) and lack a well developed CICR mechanism, which is a prerequisite for functional coupling with the host myocardium. However, it is not clear from these studies whether the immature human ES cardiomyocytes could develop functionally competent SR and CICR over a longer culture period (i.e. >3 weeks). Indeed, electrophysiologic studies on mouse ES cardiomyocytes suggest that these cells develop a functional SR and CICR mechanism in the late stages of differentiation.[112,113] In another study, 35–70% of mouse ES-derived cardiomyocytes demonstrated early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) in response to pharmacologic treatments that are known to prolong APs.[114] Both EADs and DADs are considered as underlying mechanisms for arrhythmias associated with myocardial disease. During embryonic mouse heart development (E10.5–11.5), it was shown that dual autonomic modulation of both the sympathetic and parasympathetic systems can trigger re-entry arrhythmias in the cells of the primitive

atrioventricular ring (AVR) region.[115] In contrast, both ventricular and atrial myocytes from E10.5–11.5 hearts were not susceptible to re-entry arrhythmias. Furthermore, it was suggested that the existence of such primitive AVR cells in the perinodal tissue of some adult hearts could contribute to re-entrant arrhythmia.[115] It is possible that differentiation conditions favoring development of such primitive AVR cells from ES cultures may partly explain the EADs and DADs observed by Zhang et al.[114] At present, it is not clear whether this induced arrhythmogenic potential of mouse ESderived cardiomyocytes would persist during the later stages of differentiation (i.e. >3 weeks). Taken together, these studies clearly underscore the need for the identification of mechanisms regulating proper maturation of ES-derived cardiomyocytes and also for monitoring the extent of cardiomyogenic differentiation in individual EBs. The latter part is technically challenging and perhaps can be achieved by the use of multiple, lineage-restricted, fluorescent reporters combined with live-cell imaging techniques. 5. Enrichment Methods for ES-Derived Myocardial Cells As discussed in section 1, use of undifferentiated ES cells for transplantation therapies is associated with a potential risk of teratoma development. Furthermore, the presence of undifferentiated cells in the engrafted tissue would also give rise to several undesirable cell lineages (e.g. bone, cartilage, skin). To this end, enrichment of ES-derived myocardial cells prior to transplantation is highly desirable. Enrichment strategies to date have been mainly focused on isolation of differentiated cardiomyocyte populations. Field and colleagues[42] were the first group to describe an elegant genetic selection technique for the enrichment of ES-derived

Table II. Factors increasing cardiomyogenesis of human embryonic stem (ES) cells ES-cell line (human)

Factor

Reference

H9

NGF, HGF, EGF, FGF, RA, TGFβ, activin-A

91

H1, H7, H9, H9.1, H9.2

RA, 5-aza-2′-deoxycytidine

92

HES2, HES3, HES4

Visceral endoderm-like cells

93

HES2, HES3, HES4

Serum-free medium

94

H7 and H1

IGF/PI-3-kinase/Akt signaling pathway

95

H7

Serum-free medium + CCTI

96

Miz-hES2 and HSF-6

5-azacytidine

97

H-9

Nitric oxide

98

BG01V and ReliCellhES1

BMP2 with low serum concentration

99

HUES-1 and I6

BMP2 with SU5402 (FGF receptor inhibitor)

100

H1

FBS

101

HES2, HES3, HES4

SB203580 (p38 MAP kinase inhibitor)

102

H7

Activin A, BMP4

103

BMP = bone morphogenetic protein; CCTI = creatine, carnitine, taurine, and insulin; EGF = epidermal growth factor; FBS = fetal bovine serum; FGF = fibroblast growth factor; HGF = hepatocyte growth factor; IGF = insulin-like growth factor; MAP = mitogen activated protein; NGF = nerve growth factor; PI-3-kinase = phosphatidylinositol-3-kinase; RA = retinoic acid; TGF = transforming growth factor. © 2008 Adis Data Information BV. All rights reserved.

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cardiomyocytes. In this method, undifferentiated ES cells are transfected with a dual drug resistance vector and transfected cells are selected using a hygromycin resistance gene under the control of a phosphoglycerate kinase promoter. Subsequently, the neomycin resistance gene under the control of a lineage-restricted, α cardiac myosin heavy chain (MYH6) promoter is used to select differentiated cardiomyocytes.[26,42] Similar genetic selection schemes harboring drug resistance/fluorescent reporters have been developed by other investigators to facilitate identification of cardiac progenitor cells, ventricular cells, atrial cells, and pacemaker cells.[44] Some investigators also used physical enrichment via dissection of beating areas[116] as well as percoll gradient density centrifugation[92] to enrich ES-derived cardiomyocytes. However, enrichment levels obtained by such physical methods are often less efficient than those of genetic selection methods.[42] Indeed, genetic selection via MYH6-promoted neomycin or puromycin resistance genes yielded cardiomyocyte purities of >90%.[42,117] Although initial gene transfer efforts in human ES cells appeared technically challenging (see Mummery[44]), two groups have successfully developed efficient genetic selection methods for the enrichment of human ES-derived cardiomyocytes.[105,118] Of all the enrichment methods tested to date, selection using an α cardiac MHC promoter appears to yield the highest level of myocyte purification (>90%). However, the α cardiac MYH6 promoter is active in differentiated atrial and ventricular myocytes as well as transiently active in primitive cardiomyocytes during development.[119-122] Because of this, genetic selection using this promoter was shown to enrich a heterogeneous population of cardiomyocytes.[42] In contrast, use of a myosin light chain-2v (MLC-2v) promoter for the enrichment of ES-derived cardiomyocytes was proposed to yield a homogeneous population of ventricular cardiomyocytes.[123] Given the fact that both α MYH6 and MYL2 promoters are also active in the primitive cells of the developing heart,[119-122] further refinements in selection schemes should be developed to rule out the enrichment of primitive myocardial cells with arrhythmogenic potential from ES cultures. While these genetic selection schemes are most suited to laboratory studies, it may be difficult to exploit such strategies for clinical applications. This is mainly due to the quality control issues over a long-term selection process as well as the risk of random integration of reporter genes in genetic loci controlling the vital functions of cardiomyocytes. Perhaps an ideal enrichment strategy for ES-derived myocardial cells would be screening for lineage-specific cell surface receptor expression via fluorescenceactivated cell sorting-based methodologies. Unfortunately, no unique cell-surface markers for cardiomyocytes or their progenitors have been identified. Nonetheless, recent studies have employed innovative fractionation schemes using a combination of intracellular transgene expression (e.g. NKX2-5 and ISL1) and © 2008 Adis Data Information BV. All rights reserved.

Zhang & Pasumarthi

extracellular receptors (FLK1 [KDR] and KIT) for successful enrichment of cardiac progenitor cells from mouse ES cultures.[124-126] These progenitor cells were shown to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro.[124-126] Identification of additional cell surface markers, as well as refinement of existing techniques, should facilitate further enrichment of myocardial lineage-specific fractions from ES-cell cultures in the near future. Given the structural and functional heterogeneity reported for ES-derived myocardial cells, it is important to assess whether such immature cells would fully differentiate in vivo after transplantation into diseased hearts. 6. ES-Cell Transplantation for Cardiac Repair In 1996, Field’s group[42] provided the first evidence that genetically selected mouse ES cardiomyocytes could form viable grafts when injected into the uninjured myocardium of dystrophic mice. Subsequently, multiple laboratories transplanted both undifferentiated mouse ES cells and enriched cardiomyocytes in normal as well as MI models (see table III). In some of these studies, mouse cells were injected into rat hearts with minimal or no rejection. Although some studies showed that undifferentiated ES cells could improve cardiac function with no risk of tumors (see table III), other studies clearly documented the ability of unselected ES cells to form teratomas as well as many undesired cell lineages in the heart.[30,117] The observed discrepancy between these studies could be due to the number of cells transplanted and/or differences in the analysis timepoints. Nonetheless, these studies clearly underscore the need for refinement of myocardial cell enrichment techniques from ES cultures. In 2004, Kehat et al.[54] reported the first human ES cardiomyocyte transplantation into the uninjured swine myocardium. Following this report, other investigators injected human ES cells or differentiated cardiomyocytes into the injured myocardium of different species (e.g. rat, mouse, and swine; see table IV). While some groups rationalized the use of immunocompromised nude or severe combined immunodeficiency animals for xenotransplantation studies, the absence of immune rejection in the immunocompetent recipient animals was attributed in part to the low expression or weak immunogenicity of major histocompatibility antigens in ES cells and their derivatives.[142,143] With few exceptions, the majority of ES transplantation studies in the heart utilized reporter genes such as enhanced green fluorescent protein and lacZ for tracking donor cells in the engrafted myocardium. The cardiomyocyte nature of the transplanted cells was confirmed using expression of markers such as cardiac troponin I, sarcomeric α-actin, smooth muscle α-actin, NKX2-5, NPPA (ANF), MYL7 (MLC2a), and MYL2 (MLC2v), in the engrafted areas (see tables III and IV). Consistent with the previous reports on other donor cells, transplantation of ES cardiomyocytes into normal and injured myocardium was shown to improve global myocardial function Biodrugs 2008; 22 (6)

© 2008 Adis Data Information BV. All rights reserved.

Mouse (MI)

Rat (normal)

Mouse (MI)

Mouse (normal)

Mouse (normal)

Mouse (normal)

Mouse (MI)

Mouse (MI)

Mouse (MI)

D3

D3

D3

D3

D3

D3

R1 and HM1

D3

R1

Mouse (MI)

D3

Rat (normal)

Rat (normal and MI)

D3

Sheep (MI)

Mouse (MI)

NA

CGR 8

Rat (MI)

CGR 8

NA

Mouse (MI)

Rat (MI)

CGR 8

Rat (MI)

Rat (MI)

D3

D3

Mouse (normal)

D3

D3

Host (normal or injured)

ES-cell line

Undiff

CM

Undiff

CM

Undiff

CM

Undiff

CM

Undiff

CM

CM

Undiff

Undiff

Undiff

CM

CM

CM

Undiff

CM

CM

Donor ES-cell status 7 wk 6 wk 4 wk

1 × 104 3 × 104 5 × 105

CMV-GFP α Ca. actin, MLC2v-ECFP 2 wk 3 wk 24 h to 5 wk 2 wk 2 wk 1 wk 1 mo 1, 2, 4, 8 wk 6 wk 2 wk

3 × 105 3 × 105

2 × 106 2.5 × 105 1 × 106 30 × 106 1 × 106

1 × 106 106

α Ca. actin

90 d 4 wk 2 wk

3–4 wk 8–9 wk

1.5 × 106 1.5 × 107/(ECT) 3 × 104

3 × 104–105 5 × 104

RNF4-β-Gal, CMV-EGFP α-MHC-pacIRES-EGFP A-MHC-lacZ, EF-lacZ

ND

CMV-IE-GFP

7–30 d

ND

ND

pEF1-EGFP

ND

ND

Nkx2.5-lacZ, α-actin-EYFP

ND

ND

pEF1-EGFP

ES cells labeled with SPIO

1×

6 wk

3 × 105 3 × 105

hCMVIE-GFP CMV-EGFP

ND

Analysis timepoint

No. of cells used

Reporter for cell tracking

Table III. Survey of mouse embryonic stem (ES)-cell transplantation studies

lacZ

Sarcomeric α-actin, CD45, PECAM, vWF

LacZ, GFP, sarcomeric α-actin, smooth muscle α-actin, vWF

CTnT, CTnI

GFP

CTnI, pan-cadherin, ANP, BNP

GFP

GFP, CTnI

CD3, CD4, CD8, CD45R/B220, CD11c, Mac-1, Gr-1

β-MHC, lacZ, Nkx2.5, Ki 67

EGFP, sarcomeric myosin

Sarcomeric α-actin, CD3, CD11c, MHC I, GFP

Sarcomeric α-actin, smooth muscle actin, GFP

ND

ND

α-MHC, CTnI

Troponin-1, α-MHC, GFP

MLC-2v

Sarcomeric α-actin, α-MHC, GFP, CTnI

Dystrophin

Markers used for analysis

ND

+

ND

ND

ND

+

+

ND

ND

+

ND

+

+

ND

ND

ND

+

+

ND

ND

ND

ND

ND

+

ND

ND

ND

+

ND

ND

ND

ND

ND

ND

ND

+

+

ND

ND

ND

Cx43 Angiogenesis expression

Echo

139

117

138

55

137

136

135

134

133

72

132

68

131

130

129

128

63

62

127

42

Reference

Continued next page

EP, MEA

ND

ND

Hemodyn

ND

Echo

Hemodyn

ND

Echo

ND

Echo

Echo

MRI

ECG, Echo

Hemodyn, echo

Echo

Echo

Hemodyn, echo

ND

Assay used to measure functional improvement

ES-Cell Transplantation and Myocardial Repair 367

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© 2008 Adis Data Information BV. All rights reserved.

α Ca. actin = α cardiac actin; α-MHC = α-myosin heavy chain; ANP = atrial natriuretic peptide; BNP = B-type natriuretic peptide; CFP = cyan fluorescent protein; CM = cardiomyocytes (in some cases enrichment was not performed); CMV = cytomegalovirus; CMV-IE = cytomegalovirus immediate early enhancer; CTnI = cardiac troponin I; CTnT = cardiac troponin T; Cx43 = connexin 43; echo = echocardiography; ECFP = enhanced cyan flourescent protein; ECT = engineered cardiac tissue; EF1a = elongation factor 1a promoter; EGFP = enhanced green fluorescent protein; EP = electrophysiology; EYFP = enhanced yellow fluorescent protein; GFP = green fluorescent protein; hemodyn = hemodynamics; IRES = internal ribosomal entering site; MEA = micro-electrode arrays; MHC I = major histocompatibility complex class I; MI = myocardial infarction; MLC-2v = myosin light chain-2v; MRI = magnetic resonance imaging; NA = not available; ND = not determined; PECAM = platelet/endothelial cell adhesion molecule; RNF4 = ring finger protein 4; SPIO = paramagnetic iron oxide particles; undiff = undifferentiated; vWF = Von Willebrand factor.

88 ND Mouse (normal and MI) CGR 8

Undiff or CM

ND

3 × 105–106

ND ND CFP, α-actinin

Echo

141 ND 2 wk Mouse (MI) R1 and HM1

Undiff

RNF4-lacZ, EGFP

3 × 104

ND ND ANP, sarcomeric cardiac α-actin

ND 3 wk Mouse (normal and MI) R1; CGR 8; C57

Undiff

pCX (β-actin)EGFP

5 × 105

ND ND GFP; α-fetoprotein, sarcomeric α-actin, smooth muscle α-actin, neurofilament, MY-32, CD45

Assay used to measure functional improvement Cx43 Angiogenesis expression Markers used for analysis Analysis timepoint No. of cells used Reporter for cell tracking Donor ES-cell status Host (normal or injured) ES-cell line

Table III. Contd

140

Zhang & Pasumarthi

Reference

368

using a wide range of techniques such as echocardiography, ECG, MRI, millar pressure-volume catheters, and microelectrode arrays. Analysis timepoints varied anywhere between 24 hours and 3 months post cell transplantation (tables III and IV). One obvious hurdle in this field is the sustained viability of donor cells in the engrafted myocardium.[151] A recent study from Murry and colleagues[103] found that pre-treatment of human ES cardiomyocytes with prosurvival agents increased cell survival in the infarcts as well as improving myocardial function 4 weeks after transplantation. Although a similar result was obtained by Mummery’s group using untreated human ES cardiomyocytes, the functional improvement observed at the 4-week timepoint was not apparent 12 weeks after transplantation.[148] In this regard, a number of scenarios have been discussed in the accompanying editorials, including the possibility of differences in cardiomyocyte content of the starting cell preparations.[152,153] To circumvent a possible decrease in the graft size over time, use of ES-derived cardiac precursor cells,[124-126] which may have a higher proliferative potential, has been proposed.[9,153] Given the potential of ES-derived cardiac progenitors to yield a heterogeneous group of cells, caution should be exercised in the assessment of their differentiation potential and Ca2+ handling status following intra-cardiac transplantation. Although a mechanistic basis for functional improvement following cell transplantation studies is not well understood, multiple possibilities have been proposed. Some possible mechanisms include donor cell-mediated increases in cell-to-cell coupling, angiogenesis, or paracrine pathways triggering a favorable remodeling process.[9,153] With regard to ES cardiomyocyte transplantation, some studies documented increases in angiogenesis of the engrafted myocardium. Furthermore, expression of connexin 43 (Cx43) was also observed in the membrane junctions between adjacent donor and host cardiomyocytes, indicating a potential for functional coupling (see tables III and IV). Indeed, intra-cardiac transplantation of Cx43-expressing embryonic cardiomyocytes (E14.5–16.5) was shown to prevent post-infarct arrhythmias in experimental models.[13] While it is tempting to speculate that ESderived cardiomyocytes may also suppress the arrhythmogenic risk of infarcted myocardium in vivo, caution should be exercised given the documentation of abnormal electrophysiologic behavior and immature Ca2+ handling properties of ES cardiomyocytes in vitro.[45,114] Further studies are required to confirm functional coupling between transplanted ES-derived cardiomyocytes and the host myocardium using well established techniques such as three-dimensional electrophysiologic mapping and multiphotonand Ca2+ indicator-based imaging techniques in situ or invivo.[13,16,54] Biodrugs 2008; 22 (6)

© 2008 Adis Data Information BV. All rights reserved.

Rat (MI)

Rat (MI)

HUSE-1; I6

H 9.2; I6

(normal)

Mouse

H1

Analysis timepoint

HLA-DR, slow/fast

muscle α-actin

troponin smooth

3–4 wk

EBs, CM

0.5–1 × 106

MHC, actinin,

ND

4–8 d

Undiff;

A/C, ANP

beating

Anti-human β-MHC,

3 × 106

2 mo

+

+

+

+

ND

+

ND

ND

ND

ND

ND

+

ND

Cx43 Angiogenesis expression

CTnI, Pan-cadherin, +

α-actin

GFP, sarcomeric

ANP, PCNA

7–28 d

3 wk

EBs

100 beating

106

GFP

fast skeletal MHC

S-100 protein,

β-III-tubulin,

anti-human lamin

ND

ND

ND

48–72 h

pan-cytokeratins,

targets

ND

ANP, MLC-2v,

pancentromeric

Lenti-CAG-GFP

α-actin, N-cadherin,

Alu repeat,

α-fetoprotein,

smooth muscle

Sarcomeric α-actin, β-MHC, α-MHC,

4 wk

α-actin

CTnI, sarcomeric

mitochondria Ab,

Anti-human

Markers used for analysis

Y-chromosome,

0.5–10 × 106

EBs

40–150 beating 1–3 wk

No. of cells used

human probes:

ISH with

ND

Reporter for cell tracking

(EBs), no

Undiff

CM

CM

CM

(normal)

Mouse (MI)

Pacemaker

CM

Guinea pig

H7

H1

(normal)

Rat

EBs

(normal)

H1 and H7

Beating

Swine

H9.2

Donor ES-cell status

Host (normal or injured)

ES-cell line

Table IV. Survey of human embryonic stem (ES)-cell transplantation studies

Echo

ND

ND

MRI

147

100

53

146

145

144

54

Reference

Continued next page

in vitro MEA

AP mapping,

In vivo optical

ND

mapping

Electroanatomic

Assay used to measure functional improvement

ES-Cell Transplantation and Myocardial Repair 369

Biodrugs 2008; 22 (6)

© 2008 Adis Data Information BV. All rights reserved.

Rat (MI)

Mouse (MI)

Rat (MI)

Mouse

H7

HES3-GFP

HES3-GFP

H1

Analysis timepoint

2–3 d

actin, MLC-2a,

timepoints

desmoplakin 1 and

Pan-cadherin,

MF20, cytokeratin

α-actinin, α-MHC,

tropomyosin,

GFP, sarcomeric

Various

laminin

desmoplakin,

muscle α-actin,

Ki 67, smooth

MLC-2a, MLC-2v,

troma 1, PECAM-1,

tropomyosin,

GFP, α-actinin,

mitochondrial Ab,

ND

+

ND

ND

ND

ND

measurement

Force

ND

MRI

Echo, MRI

Assay used to measure functional improvement

150

149

148

103

Reference

nuclear antigen; PECAM-1 = platelet/endothelial cell adhesion molecule-1; undiff = undifferentiated; + indicates positive.

myocardial infarction; MLC-2a = myosin light chain-2a; MLC-2v = myosin light chain-2v; MRI = magnetic resonance imaging; ND = not determined; PCNA = proliferating cell

troponin I; Cx43 = connexin 43; EBs = embryoid bodies; echo = echocardiography; GFP = green fluorescent protein; ISH = in situ hybridization; MHC = myosin heavy chain; MI =

Ab = antibody; Alu repeat = short repetitive elements defined by Alu enzyme; ANP = atrial natriuretic peptide; AP = action potential; CM = cardiomyocytes; CTnI = cardiac

mitochondria

α-actinin, human

clusters

3–10 beating

2 × 106

ND

Cx43 Angiogenesis expression

Anti-human nuclear/ +

Nkx 2.5, MLC-2v

CTnI, human

pan-cadherin,

MHC, β-MHC,

Sarcomeric

Markers used for analysis

slices)

ND

ND

probe

12 wk

pancentromeric

2 d, 1 wk, 3 wk, 10 wk,

0.2–1 × 106

3.9 ± 0.8 × 107 4 wk

No. of cells used

human-specific

ISH with a

ND

Reporter for cell tracking

2, vimentin, cardiac

CM

CM

CM

CM

Donor ES-cell status

ventricular

(ischemic

Host (normal or injured)

ES-cell line

Table IV. Contd

370 Zhang & Pasumarthi

Biodrugs 2008; 22 (6)

ES-Cell Transplantation and Myocardial Repair

7. Conclusion and Future Perspectives Recent studies suggest that transplantation of donor cells in diseased myocardium can improve myocardial function over a short period of time (1–4 weeks). While long-term follow-up studies are warranted, it is generally perceived that the beneficial effects of transplanted cells are mainly due to increased angiogenesis or favorable scar remodeling in the engrafted myocardium. With the exception of fetal cardiomyocytes, other donor cells are incapable of electrical coupling with the host myocardium. In this regard, ES cells serve as an attractive source for the generation of unlimited quantities of cardiomyocytes for myocardial repair. The initial success in the application of ES cells to partially repair and improve myocardial function in experimental models of heart disease has been quite promising. However, multiple hurdles need to be crossed before the potential benefits of ES cells can be translated to the clinic. Given the heterogeneity of Ca2+ handling proteins and the electrophysiologic properties of ES-derived cardiomyocytes, current enrichment methods should be further refined to derive a homogenous population of ES cardiomyocytes. In addition, electrical coupling of ES-derived cardiomyocytes with the host myocardium is yet to be confirmed at the cellular level. This is particularly important in view of the increased arrhythmogenic risk seen with skeletal myoblast transplantation in phase I clinical trials. An urgent requirement in this field is to perform long-term monitoring for the incidence of arrhythmias and teratomas in animals transplanted with ES cardiomyocyte preparations. A more cautious approach to the validation of ES cardiomyocytes in experimental models would certainly prevent future disappointments as seen with skeletal myoblast studies. In addition, future studies should focus on optimizing the yield of cardiomyocytes from ES-cell cultures as well as developing ethically approved methods for derivation of patient-specific ES-cell lines. In view of our initial progress in the validation of ES-derived cardiomycoytes as a potential donor cell type, we should have an optimistic mindset to sail across the hurdles toward an ultimate clinical realization. Acknowledgments We apologize to those whose work could not be cited because of space limitations. We thank the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada for support. Dr Pasumarthi is a New Investigator of the Heart and Stroke Foundation of Canada. We thank Karim Wafa, Sarah Remley, and Adam Hotchkiss (members of the Pasumarthi laboratory) for their helpful comments on this manuscript. The authors have no conflicts of interest that are directly relevant to the content of this review.

References 1. Pasumarthi KB, Field LJ. Cardiomyocyte cell cycle regulation. Circ Res 2002; 90 (10): 1044-54 © 2008 Adis Data Information BV. All rights reserved.

371

2. McMullen NM, Gaspard GJ, Pasmarthi KB. Reactivation of cardiomyocyte cell cycle: a potential approach for myocardial regeneration. Signal Transduction 2005; 5 (3): 126-41 3. Statistics Canada. Causes of death 2002. Ottawa: Statistics Canada, 2004 [online]. Available from URL: http://www.statcan.ca/english/freepub/84-208-XIE/84208-XIE2004002.htm [Accessed 2008 Jul 28] 4. Lloyd-Jones DM. The risk of congestive heart failure: sobering lessons from the Framingham Heart Study. Curr Cardiol Rep 2001; 3 (3): 184-90 5. Guyatt GH, Devereaux PJ. A review of heart failure treatment. Mt Sinai J Med 2004; 71 (1): 47-54 6. Braunwald E, Antman EM. Evidence-based coronary care. Ann Intern Med 1997; 126 (7): 551-3 7. Bardy GH, Lee KL, Mark DB, et al. Amiodarone or an implantable cardioverterdefibrillator for congestive heart failure. N Engl J Med 2005; 352 (3): 225-37 8. Rose EA, Gelijns AC, Moskowitz AJ, et al. Long-term mechanical left ventricular assistance for end-stage heart failure. N Engl J Med 2001; 345 (20): 1435-43 9. McMullen NM, Pasumarthi KB. Donor cell transplantation for myocardial disease: does it complement current pharmacological therapies? Can J Physiol Pharmacol 2007; 85 (1): 1-15 10. Hagege AA, Marolleau JP, Vilquin JT, et al. Skeletal myoblast transplantation in ischemic heart failure: long-term follow-up of the first phase I cohort of patients. Circulation 2006; 114 (1 Suppl.): I108-13 11. Balsam LB, Robbins RC. Haematopoietic stem cells and repair of the ischaemic heart. Clin Sci (Lond) 2005; 109 (6): 483-92 12. Murry CE, Field LJ, Menasche P. Cell-based cardiac repair: reflections at the 10year point. Circulation 2005; 112 (20): 3174-83 13. Roell W, Lewalter T, Sasse P, et al. Engraftment of connexin 43-expressing cells prevents post-infarct arrhythmia. Nature 2007; 450 (7171): 819-24 14. Li RK, Weisel RD, Mickle DA, et al. Autologous porcine heart cell transplantation improved heart function after a myocardial infarction. J Thorac Cardiovasc Surg 2000; 119 (1): 62-8 15. Leor J, Patterson M, Quinones MJ, et al. Transplantation of fetal myocardial tissue into the infarcted myocardium of rat: a potential method for repair of infarcted myocardium? Circulation 1996; 94 (9 Suppl.): II332-6 16. Rubart M, Pasumarthi KB, Nakajima H, et al. Physiological coupling of donor and host cardiomyocytes after cellular transplantation. Circ Res 2003; 92 (11): 1217-24 17. Etzion S, Battler A, Barbash IM, et al. Influence of embryonic cardiomyocyte transplantation on the progression of heart failure in a rat model of extensive myocardial infarction. J Mol Cell Cardiol 2001; 33 (7): 1321-30 18. Sakakibara Y, Nishimura K, Tambara K, et al. Prevascularization with gelatin microspheres containing basic fibroblast growth factor enhances the benefits of cardiomyocyte transplantation. J Thorac Cardiovasc Surg 2002; 124 (1): 50-6 19. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981; 78 (12): 7634-8 20. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292 (5819): 154-6 21. Doetschman T, Williams P, Maeda N. Establishment of hamster blastocyst-derived embryonic stem (ES) cells. Dev Biol 1988; 127 (1): 224-7 22. Wheeler MB. Development and validation of swine embryonic stem cells: a review. Reprod Fertil Dev 1994; 6 (5): 563-8 23. Prelle K, Vassiliev IM, Vassilieva SG, et al. Establishment of pluripotent cell lines from vertebrate species: present status and future prospects. Cells Tissues Organs 1999; 165 (3-4): 220-36 24. Thomson JA, Kalishman J, Golos TG, et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 1995; 92 (17): 7844-8 25. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts [published erratum appears in: Science 1998; 282 (5395): 1827]. Science 1998; 282 (5391): 1145-7 26. Pasumarthi KB, Field LJ. Cardiomyocyte enrichment in differentiating ES cell cultures: strategies and applications. Methods Mol Biol 2002; 185: 157-68 27. Nichols J, Evans EP, Smith AG. Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity. Development 1990; 110 (4): 1341-8 28. Amit M, Itskovitz-Eldor J. Feeder-free culture of human embryonic stem cells. Methods Enzymol 2006; 420: 37-49 29. Doetschman TC, Eistetter H, Katz M, et al. The in vitro development of blastocystderived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 1985; 87: 27-45 Biodrugs 2008; 22 (6)

372

30. Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol 2005; 23 (7): 845-56 31. Das S, Bonaguidi M, Muro K, et al. Generation of embryonic stem cells: limitations of and alternatives to inner cell mass harvest. Neurosurg Focus 2008; 24 (3-4): E4 32. Pralong D, Mrozik K, Occhiodoro F, et al. A novel method for somatic cell nuclear transfer to mouse embryonic stem cells. Cloning Stem Cells 2005; 7 (4): 265-71 33. Tamada H, Kikyo N. Nuclear reprogramming in mammalian somatic cell nuclear cloning. Cytogenet Genome Res 2004; 105 (2-4): 285-91 34. Klimanskaya I, Chung Y, Becker S, et al. Human embryonic stem cell lines derived from single blastomeres. Nature 2006; 444 (7118): 481-5 35. Chung Y, Klimanskaya I, Becker S, et al. Embryonic and extra-embryonic stem cell lines derived from single mouse blastomeres. Nature 2006; 439 (7073): 216-9 36. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131 (5): 861-72 37. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318 (5858): 1917-20 38. Park IH, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008; 451 (7175): 141-6 39. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126 (4): 663-76 40. Pera MF. Stem cells: a new year and a new era. Nature 2008; 451 (7175): 135-6 41. Kehat I, Kenyagin-Karsenti D, Snir M, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001; 108 (3): 407-14 42. Klug MG, Soonpaa MH, Koh GY, et al. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J Clin Invest 1996; 98 (1): 216-24 43. Wobus AM, Wallukat G, Hescheler J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 1991; 48 (3): 173-82 44. Mummery C. Genetic selection of cardiomyocytes from human embryonic stem cells. Mol Ther 2007; 15 (11): 1908-9 45. Siu CW, Moore JC, Li RA. Human embryonic stem cell-derived cardiomyocytes for heart therapies. Cardiovasc Hematol Disord Drug Targets 2007; 7 (2): 14552 46. Boheler KR, Czyz J, Tweedie D, et al. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res 2002; 91 (3): 189-201 47. Maltsev VA, Wobus AM, Rohwedel J, et al. Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. Circ Res 1994; 75 (2): 233-44 48. Mummery C, Ward D, van den Brink CE, et al. Cardiomyocyte differentiation of mouse and human embryonic stem cells. J Anat 2002; 200 (Pt 3): 233-42 49. Muller M, Fleischmann BK, Selbert S, et al. Selection of ventricular-like cardiomyocytes from ES cells in vitro. Faseb J 2000; 14 (15): 2540-8 50. Wobus AM, Kaomei G, Shan J, et al. Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes. J Mol Cell Cardiol 1997; 29 (6): 1525-39 51. Hescheler J, Fleischmann BK, Lentini S, et al. Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. Cardiovasc Res 1997; 36 (2): 149-62 52. Banach K, Halbach MD, Hu P, et al. Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells. Am J Physiol Heart Circ Physiol 2003; 284 (6): H2114-23 53. Cui L, Johkura K, Takei S, et al. Structural differentiation, proliferation, and association of human embryonic stem cell-derived cardiomyocytes in vitro and in their extracardiac tissues. J Struct Biol 2007; 158 (3): 307-17 54. Kehat I, Khimovich L, Caspi O, et al. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol 2004; 22 (10): 1282-9 55. Guo XM, Zhao YS, Chang HX, et al. Creation of engineered cardiac tissue in vitro from mouse embryonic stem cells. Circulation 2006; 113 (18): 2229-37 56. Naito H, Melnychenko I, Didie M, et al. Optimizing engineered heart tissue for therapeutic applications as surrogate heart muscle. Circulation 2006; 114 (1 Suppl.): I72-8 © 2008 Adis Data Information BV. All rights reserved.

Zhang & Pasumarthi

57. Wang X, Wei G, Yu W, et al. Scalable producing embryoid bodies by rotary cell culture system and constructing engineered cardiac tissue with ES-derived cardiomyocytes in vitro. Biotechnol Prog 2006; 22 (3): 811-8 58. Bauwens C, Yin T, Dang S, et al. Development of a perfusion fed bioreactor for embryonic stem cell-derived cardiomyocyte generation: oxygen-mediated enhancement of cardiomyocyte output. Biotechnol Bioeng 2005; 90 (4): 452-61 59. Schroeder M, Niebruegge S, Werner A, et al. Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control. Biotechnol Bioeng 2005; 92 (7): 920-33 60. Zandstra PW, Bauwens C, Yin T, et al. Scalable production of embryonic stem cell-derived cardiomyocytes. Tissue Eng 2003; 9 (4): 767-78 61. Sauer H, Rahimi G, Hescheler J, et al. Role of reactive oxygen species and phosphatidylinositol 3-kinase in cardiomyocyte differentiation of embryonic stem cells. FEBS Lett 2000; 476 (3): 218-23 62. Behfar A, Zingman LV, Hodgson DM, et al. Stem cell differentiation requires a paracrine pathway in the heart. Faseb J 2002; 16 (12): 1558-66 63. Yang Y, Min JY, Rana JS, et al. VEGF enhances functional improvement of postinfarcted hearts by transplantation of ESC-differentiated cells. J Appl Physiol 2002; 93 (3): 1140-51 64. Dell’Era P, Ronca R, Coco L, et al. Fibroblast growth factor receptor-1 is essential for in vitro cardiomyocyte development. Circ Res 2003; 93 (5): 414-20 65. Hidaka K, Lee JK, Kim HS, et al. Chamber-specific differentiation of Nkx2.5positive cardiac precursor cells from murine embryonic stem cells. Faseb J 2003; 17 (6): 740-2 66. Sachinidis A, Gissel C, Nierhoff D, et al. Identification of platelet-derived growth factor-BB as cardiogenesis-inducing factor in mouse embryonic stem cells under serum-free conditions. Cell Physiol Biochem 2003; 13 (6): 423-9 67. Rudy-Reil D, Lough J. Avian precardiac endoderm/mesoderm induces cardiac myocyte differentiation in murine embryonic stem cells. Circ Res 2004; 94 (12): e107-16 68. Kofidis T, de Bruin JL, Yamane T, et al. Insulin-like growth factor promotes engraftment, differentiation, and functional improvement after transfer of embryonic stem cells for myocardial restoration. Stem Cells 2004; 22 (7): 1239-45 69. Kanno S, Kim PK, Sallam K, et al. Nitric oxide facilitates cardiomyogenesis in mouse embryonic stem cells. Proc Natl Acad Sci U S A 2004; 101 (33): 1227781 70. Kawamura T, Ono K, Morimoto T, et al. Acetylation of GATA-4 is involved in the differentiation of embryonic stem cells into cardiac myocytes. J Biol Chem 2005; 280 (20): 19682-8 71. Honda M, Hamazaki TS, Komazaki S, et al. RXR agonist enhances the differentiation of cardiomyocytes derived from embryonic stem cells in serum-free conditions. Biochem Biophys Res Commun 2005; 333 (4): 1334-40 72. Menard C, Hagege AA, Agbulut O, et al. Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium: a preclinical study. Lancet 2005; 366 (9490): 1005-12 73. Singla DK, Sun B. Transforming growth factor-beta2 enhances differentiation of cardiac myocytes from embryonic stem cells. Biochem Biophys Res Commun 2005; 332 (1): 135-41 74. Czyz J, Guan K, Zeng Q, et al. Loss of beta 1 integrin function results in upregulation of connexin expression in embryonic stem cell-derived cardiomyocytes. Int J Dev Biol 2005; 49 (1): 33-41 75. Sachinidis A, Schwengberg S, Hippler-Altenburg R, et al. Identification of small signalling molecules promoting cardiac-specific differentiation of mouse embryonic stem cells. Cell Physiol Biochem 2006; 18 (6): 303-14 76. Zhu DY, Lou YJ. Icariin-mediated expression of cardiac genes and modulation of nitric oxide signaling pathway during differentiation of mouse embryonic stem cells into cardiomyocytes in vitro. Acta Pharmacol Sin 2006; 27 (3): 311-20 77. Bin Z, Sheng LG, Gang ZC, et al. Efficient cardiomyocyte differentiation of embryonic stem cells by bone morphogenetic protein-2 combined with visceral endoderm-like cells. Cell Biol Int 2006; 30 (10): 769-76 78. Chen Y, Shao JZ, Xiang LX, et al. Cyclic adenosine 3′,5′-monophosphate induces differentiation of mouse embryonic stem cells into cardiomyocytes. Cell Biol Int 2006; 30 (4): 301-7 79. E LL, Zhao YS, Guo XM, et al. Enrichment of cardiomyocytes derived from mouse embryonic stem cells. J Heart Lung Transplant 2006; 25 (6): 664-74 80. Ding L, Liang X, Zhu D, et al. Peroxisome proliferator-activated receptor alpha is involved in cardiomyocyte differentiation of murine embryonic stem cells in vitro. Cell Biol Int 2007; 31 (9): 1002-9 Biodrugs 2008; 22 (6)

ES-Cell Transplantation and Myocardial Repair

81. Singh AM, Li FQ, Hamazaki T, et al. Chibby, an antagonist of the Wnt/betacatenin pathway, facilitates cardiomyocyte differentiation of murine embryonic stem cells. Circulation 2007; 115 (5): 617-26 82. Hatami L, Valojerdi MR, Mowla SJ. Effects of oxytocin on cardiomyocyte differentiation from mouse embryonic stem cells. Int J Cardiol 2007; 117 (1): 80-9 83. Khezri S, Valojerdi MR, Sepehri H, et al. Effect of basic fibroblast growth factor on cardiomyocyte differentiation from mouse embryonic stem cells. Saudi Med J 2007; 28 (2): 181-6 84. Taha MF, Valojerdi MR, Mowla SJ. Effect of bone morphogenetic protein-4 (BMP-4) on cardiomyocyte differentiation from mouse embryonic stem cell. Int J Cardiol 2007; 120 (1): 92-101 85. Hofner M, Hollrigl A, Puz S, et al. Desmin stimulates differentiation of cardiomyocytes and up-regulation of brachyury and nkx2.5. Differentiation 2007; 75 (7): 605-15 86. Roggia C, Ukena C, Bohm M, et al. Hepatocyte growth factor (HGF) enhances cardiac commitment of differentiating embryonic stem cells by activating PI3 kinase. Exp Cell Res 2007; 313 (5): 921-30 87. Bugorsky R, Perriard JC, Vassalli G. N-cadherin is essential for retinoic acidmediated cardiomyogenic differentiation in mouse embryonic stem cells. Eur J Histochem 2007; 51 (3): 181-92 88. Behfar A, Perez-Terzic C, Faustino RS, et al. Cardiopoietic programming of embryonic stem cells for tumor-free heart repair. J Exp Med 2007; 204 (2): 40520 89. Kim HS, Cho JW, Hidaka K, et al. Activation of MEK-ERK by heregulin-beta1 promotes the development of cardiomyocytes derived from ES cells. Biochem Biophys Res Commun 2007; 361 (3): 732-8 90. Gassanov N, Devost D, Danalache B, et al. Functional activity of the carboxylterminally extended oxytocin precursor peptide during cardiac differentiation of embryonic stem cells. Stem Cells 2008; 26 (1): 45-54 91. Schuldiner M, Yanuka O, Itskovitz-Eldor J, et al. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2000; 97 (21): 11307-12 92. Xu C, Police S, Rao N, et al. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res 2002; 91 (6): 501-8 93. Mummery C, Ward-van Oostwaard D, Doevendans P, et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 2003; 107 (21): 2733-40 94. Passier R, Oostwaard DW, Snapper J, et al. Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures. Stem Cells 2005; 23 (6): 772-80 95. McDevitt TC, Laflamme MA, Murry CE. Proliferation of cardiomyocytes derived from human embryonic stem cells is mediated via the IGF/PI 3-kinase/Akt signaling pathway. J Mol Cell Cardiol 2005; 39 (6): 865-73 96. Xu C, He JQ, Kamp TJ, et al. Human embryonic stem cell-derived cardiomyocytes can be maintained in defined medium without serum. Stem Cells Dev 2006; 15 (6): 931-41 97. Yoon BS, Yoo SJ, Lee JE, et al. Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5azacytidine treatment. Differentiation 2006; 74 (4): 149-59 98. Mujoo K, Krumenacker JS, Wada Y, et al. Differential expression of nitric oxide signaling components in undifferentiated and differentiated human embryonic stem cells. Stem Cells Dev 2006; 15 (6): 779-87 99. Pal R, Khanna A. Similar pattern in cardiac differentiation of human embryonic stem cell lines, BG01V and ReliCellhES1, under low serum concentration supplemented with bone morphogenetic protein-2. Differentiation 2007; 75 (2): 112-22 100. Tomescot A, Leschik J, Bellamy V, et al. Differentiation in vivo of cardiac committed human embryonic stem cells in postmyocardial infarcted rats. Stem Cells 2007; 25 (9): 2200-5 101. Bettiol E, Sartiani L, Chicha L, et al. Fetal bovine serum enables cardiac differentiation of human embryonic stem cells. Differentiation 2007; 75 (8): 669-81 102. Graichen R, Xu X, Braam SR, et al. Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK. Differentiation 2008; 76 (4): 357-70 103. Laflamme MA, Chen KY, Naumova AV, et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol 2007; 25 (9): 1015-24 © 2008 Adis Data Information BV. All rights reserved.

373

104. He JQ, Ma Y, Lee Y, et al. Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circ Res 2003; 93 (1): 32-9 105. Anderson D, Self T, Mellor IR, et al. Transgenic enrichment of cardiomyocytes from human embryonic stem cells. Mol Ther 2007; 15 (11): 2027-36 106. Reppel M, Boettinger C, Hescheler J. Beta-adrenergic and muscarinic modulation of human embryonic stem cell-derived cardiomyocytes. Cell Physiol Biochem 2004; 14 (4-6): 187-96 107. Fijnvandraat AC, van Ginneken AC, de Boer PA, et al. Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube. Cardiovasc Res 2003; 58 (2): 399-409 108. Fijnvandraat AC, van Ginneken AC, Schumacher CA, et al. Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium. J Mol Cell Cardiol 2003; 35 (12): 1461-72 109. Dolnikov K, Shilkrut M, Zeevi-Levin N, et al. Functional properties of human embryonic stem cell-derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction. Stem Cells 2006; 24 (2): 236-45 110. Dolnikov K, Shilkrut M, Zeevi-Levin N, et al. Functional properties of human embryonic stem cell-derived cardiomyocytes. Ann N Y Acad Sci 2005; 1047: 66-75 111. Liu J, Fu JD, Siu CW, et al. Functional sarcoplasmic reticulum for calcium handling of human embryonic stem cell-derived cardiomyocytes: insights for driven maturation. Stem Cells 2007; 25 (12): 3038-44 112. Fu JD, Li J, Tweedie D, et al. Crucial role of the sarcoplasmic reticulum in the developmental regulation of Ca2+ transients and contraction in cardiomyocytes derived from embryonic stem cells. Faseb J 2006; 20 (1): 181-3 113. Fu JD, Yu HM, Wang R, et al. Developmental regulation of intracellular calcium transients during cardiomyocyte differentiation of mouse embryonic stem cells. Acta Pharmacol Sin 2006; 27 (7): 901-10 114. Zhang YM, Hartzell C, Narlow M, et al. Stem cell-derived cardiomyocytes demonstrate arrhythmic potential. Circulation 2002; 106 (10): 1294-9 115. Valderrabano M, Chen F, Dave AS, et al. Atrioventricular ring reentry in embryonic mouse hearts. Circulation 2006; 114 (6): 543-9 116. Segev H, Kenyagin-Karsenti D, Fishman B, et al. Molecular analysis of cardiomyocytes derived from human embryonic stem cells. Dev Growth Differ 2005; 47 (5): 295-306 117. Kolossov E, Bostani T, Roell W, et al. Engraftment of engineered ES cell-derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium. J Exp Med 2006; 203 (10): 2315-27 118. Huber I, Itzhaki I, Caspi O, et al. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation. Faseb J 2007; 21 (10): 255163 119. Lyons GE, Ontell M, Cox R, et al. The expression of myosin genes in developing skeletal muscle in the mouse embryo. J Cell Biol 1990; 111 (4): 1465-76 120. Franco D, Markman MM, Wagenaar GT, et al. Myosin light chain 2a and 2v identifies the embryonic outflow tract myocardium in the developing rodent heart. Anat Rec 1999; 254 (1): 135-46 121. Ng WA, Grupp IL, Subramaniam A, et al. Cardiac myosin heavy chain mRNA expression and myocardial function in the mouse heart. Circ Res 1991; 68 (6): 1742-50 122. Siedner S, Kruger M, Schroeter M, et al. Developmental changes in contractility and sarcomeric proteins from the early embryonic to the adult stage in the mouse heart. J Physiol 2003; 548 (Pt 2): 493-505 123. Nir SG, David R, Zaruba M, et al. Human embryonic stem cells for cardiovascular repair. Cardiovasc Res 2003; 58 (2): 313-23 124. Moretti A, Caron L, Nakano A, et al. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification. Cell 2006; 127 (6): 1151-65 125. Wu SM, Fujiwara Y, Cibulsky SM, et al. Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart. Cell 2006; 127 (6): 1137-50 126. Kattman SJ, Huber TL, Keller GM. Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages. Dev Cell 2006; 11 (5): 723-32 127. Min JY, Yang Y, Converso KL, et al. Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats. J Appl Physiol 2002; 92 (1): 288-96 Biodrugs 2008; 22 (6)

374

Zhang & Pasumarthi

128. Min JY, Yang Y, Sullivan MF, et al. Long-term improvement of cardiac function in rats after infarction by transplantation of embryonic stem cells. J Thorac Cardiovasc Surg 2003; 125 (2): 361-9

142. Drukker M, Katchman H, Katz G, et al. Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells 2006; 24 (2): 221-9

129. Hodgson DM, Behfar A, Zingman LV, et al. Stable benefit of embryonic stem cell therapy in myocardial infarction. Am J Physiol Heart Circ Physiol 2004; 287 (2): H471-9

143. Tian L, Catt JW, O’Neill C, et al. Expression of immunoglobulin superfamily cell adhesion molecules on murine embryonic stem cells. Biol Reprod 1997; 57 (3): 561-8

130. Himes N, Min JY, Lee R, et al. In vivo MRI of embryonic stem cells in a mouse model of myocardial infarction. Magn Reson Med 2004; 52 (5): 1214-9

144. Laflamme MA, Gold J, Xu C, et al. Formation of human myocardium in the rat heart from human embryonic stem cells. Am J Pathol 2005; 167 (3): 663-71

131. Kofidis T, de Bruin JL, Hoyt G, et al. Injectable bioartificial myocardial tissue for large-scale intramural cell transfer and functional recovery of injured heart muscle. J Thorac Cardiovasc Surg 2004; 128 (4): 571-8

145. Xue T, Cho HC, Akar FG, et al. Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers. Circulation 2005; 111 (1): 11-20

132. Naito H, Nishizaki K, Yoshikawa M, et al. Xenogeneic embryonic stem cellderived cardiomyocyte transplantation. Transplant Proc 2004; 36 (8): 2507-8 133. Swijnenburg RJ, Tanaka M, Vogel H, et al. Embryonic stem cell immunogenicity increases upon differentiation after transplantation into ischemic myocardium. Circulation 2005; 112 (9 Suppl.): I166-72 134. Min JY, Huang X, Xiang M, et al. Homing of intravenously infused embryonic stem cell-derived cells to injured hearts after myocardial infarction. J Thorac Cardiovasc Surg 2006; 131 (4): 889-97 135. Kofidis T, de Bruin JL, Yamane T, et al. Stimulation of paracrine pathways with growth factors enhances embryonic stem cell engraftment and host-specific differentiation in the heart after ischemic myocardial injury. Circulation 2005; 111 (19): 2486-93 136. Johkura K, Cui L, Yue F, et al. Natriuretic peptides in ectopic myocardial tissues originating from mouse embryonic stem cells. Microsc Res Tech 2005; 66 (4): 165-72 137. Malek S, Kaplan E, Wang JF, et al. Successful implantation of intravenously administered stem cells correlates with severity of inflammation in murine myocarditis. Pflugers Arch 2006; 452 (3): 268-75 138. Singla DK, Hacker TA, Ma L, et al. Transplantation of embryonic stem cells into the infarcted mouse heart: formation of multiple cell types. J Mol Cell Cardiol 2006; 40 (1): 195-200 139. Nelson TJ, Ge ZD, Van Orman J, et al. Improved cardiac function in infarcted mice after treatment with pluripotent embryonic stem cells. Anat Rec A Discov Mol Cell Evol Biol 2006; 288 (11): 1216-24 140. Nussbaum J, Minami E, Laflamme MA, et al. Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response. Faseb J 2007; 21 (7): 1345-57 141. Singla DK, Lyons GE, Kamp TJ. Transplanted embryonic stem cells following mouse myocardial infarction inhibit apoptosis and cardiac remodeling. Am J Physiol Heart Circ Physiol 2007; 293 (2): H1308-14

© 2008 Adis Data Information BV. All rights reserved.

146. Kofidis T, Lebl DR, Swijnenburg RJ, et al. Allopurinol/uricase and ibuprofen enhance engraftment of cardiomyocyte-enriched human embryonic stem cells and improve cardiac function following myocardial injury. Eur J Cardiothorac Surg 2006; 29 (1): 50-5 147. Leor J, Gerecht S, Cohen S, et al. Human embryonic stem cell transplantation to repair the infarcted myocardium. Heart 2007; 93 (10): 1278-84 148. van Laake LW, Passier R, Monshouwer-Kloots J, et al. Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. Stem Cell Res 2007; 1: 924 149. Dai W, Field LJ, Rubart M, et al. Survival and maturation of human embryonic stem cell-derived cardiomyocytes in rat hearts. J Mol Cell Cardiol 2007; 43 (4): 504-16 150. Pillekamp F, Reppel M, Rubenchyk O, et al. Force measurements of human embryonic stem cell-derived cardiomyocytes in an in vitro transplantation model. Stem Cells 2007; 25 (1): 174-80 151. Reinecke H, Murry CE. Taking the death toll after cardiomyocyte grafting: a reminder of the importance of quantitative biology. J Mol Cell Cardiol 2002; 34 (3): 251-3 152. Elliott DA, Harvey RP. Time to mend a broken heart. Stem Cell Res 2007; 1: 4-6 153. Rubart M, Field LJ. ES cells for troubled hearts. Nat Biotechnol 2007; 25 (9): 9934

Correspondence: Dr Kishore B.S. Pasumarthi, Department of Pharmacology, Dalhousie University, Sir Charles Tupper Building, 5850 College Street, Halifax, B3H 1X5, Canada. E-mail: [email protected]

Biodrugs 2008; 22 (6)