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Ilanit Itzhaki,
Leonid Maizels, Irit Huber,
Amira Gepstein,
Gil Arbel,
Oren Caspi,
Liron Miller,
Bernard Belhassen,
Eyal Nof,
Michael Glikson,
Lior Gepstein
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ABSTRACT: The goal of this study was to establish a patient-specific human-induced pluripotent stem cells (hiPSCs) model of catecholaminergic polymorphic ventricular tachycardia (CPVT).
CPVT is a familial arrhythmogenic syndrome characterized by abnormal calcium (Ca(2+)) handling, ventricular arrhythmias, and sudden cardiac death.
Dermal fibroblasts were obtained from a CPVT patient due to the M4109R heterozygous point RYR2 mutation and reprogrammed to generate the CPVT-hiPSCs. The patient-specific hiPSCs were coaxed to differentiate into the cardiac lineage and compared with healthy control hiPSCs-derived cardiomyocytes (hiPSCs-CMs).
Intracellular electrophysiological recordings demonstrated the development of delayed afterdepolarizations in 69% of the CPVT-hiPSCs-CMs compared with 11% in healthy control cardiomyocytes. Adrenergic stimulation by isoproterenol (1 μM) or forskolin (5 μM) increased the frequency and magnitude of afterdepolarizations and also led to development of triggered activity in the CPVT-hiPSCs-CMs. In contrast, flecainide (10 μM) and thapsigargin (10 μM) eliminated all afterdepolarizations in these cells. The latter finding suggests an important role for internal Ca(2+) stores in the pathogenesis of delayed afterdepolarizations. Laser-confocal Ca(2+) imaging revealed significant whole-cell [Ca(2+)] transient irregularities (frequent local and large-storage Ca(2+)-release events, broad and double-humped transients, and triggered activity) in the CPVT cardiomyocytes that worsened with adrenergic stimulation and Ca(2+) overload and improved with beta-blockers. Store-overload-induced Ca(2+) release was also identified in the hiPSCs-CMs and the threshold for such events was significantly reduced in the CPVT cells.
This study highlights the potential of hiPSCs for studying inherited arrhythmogenic syndromes, in general, and CPVT specifically. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care, and aid in the development of new therapies.
Journal of the American College of Cardiology 06/2012; 60(11):990-1000. · 14.16 Impact Factor
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ABSTRACT: AimsMyocardial cell replacement therapies are hampered by a paucity of sources for human cardiomyocytes and by the expected immune rejection of allogeneic cell grafts. The ability to derive patient-specific human-induced pluripotent stem cells (hiPSCs) may provide a solution to these challenges. We aimed to derive hiPSCs from heart failure (HF) patients, to induce their cardiomyocyte differentiation, to characterize the generated hiPSC-derived cardiomyocytes (hiPSC-CMs), and to evaluate their ability to integrate with pre-existing cardiac tissue.Methods and resultsDermal fibroblasts from two HF patients were reprogrammed by retroviral delivery of Oct4, Sox2, and Klf4 or by using an excisable polycistronic lentiviral vector. The resulting HF-hiPSCs displayed adequate reprogramming properties and could be induced to differentiate into cardiomyocytes with the same efficiency as control hiPSCs (derived from human foreskin fibroblasts). Gene expression and immunostaining studies confirmed the cardiomyocyte phenotype of the differentiating HF-hiPSC-CMs. Multi-electrode array recordings revealed the development of a functional cardiac syncytium and adequate chronotropic responses to adrenergic and cholinergic stimulation. Next, functional integration and synchronized electrical activities were demonstrated between hiPSC-CMs and neonatal rat cardiomyocytes in co-culture studies. Finally, in vivo transplantation studies in the rat heart revealed the ability of the HF-hiPSC-CMs to engraft, survive, and structurally integrate with host cardiomyocytes.ConclusionsHuman-induced pluripotent stem cells can be established from patients with advanced heart failure and coaxed to differentiate into cardiomyocytes, which can integrate with host cardiac tissue. This novel source for patient-specific heart cells may bring a unique value to the emerging field of cardiac regenerative medicine.
European Heart Journal 05/2012; · 10.48 Impact Factor
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Ilanit Itzhaki,
Leonid Maizels, Irit Huber,
Limor Zwi-Dantsis,
Oren Caspi,
Aaron Winterstern,
Oren Feldman,
Amira Gepstein,
Gil Arbel,
Haim Hammerman,
Monther Boulos,
Lior Gepstein
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ABSTRACT: The ability to generate patient-specific human induced pluripotent stem cells (iPSCs) offers a new paradigm for modelling human disease and for individualizing drug testing. Congenital long QT syndrome (LQTS) is a familial arrhythmogenic syndrome characterized by abnormal ion channel function and sudden cardiac death. Here we report the development of a patient/disease-specific human iPSC line from a patient with type-2 LQTS (which is due to the A614V missense mutation in the KCNH2 gene). The generated iPSCs were coaxed to differentiate into the cardiac lineage. Detailed whole-cell patch-clamp and extracellular multielectrode recordings revealed significant prolongation of the action-potential duration in LQTS human iPSC-derived cardiomyocytes (the characteristic LQTS phenotype) when compared to healthy control cells. Voltage-clamp studies confirmed that this action-potential-duration prolongation stems from a significant reduction of the cardiac potassium current I(Kr). Importantly, LQTS-derived cells also showed marked arrhythmogenicity, characterized by early-after depolarizations and triggered arrhythmias. We then used the LQTS human iPSC-derived cardiac-tissue model to evaluate the potency of existing and novel pharmacological agents that may either aggravate (potassium-channel blockers) or ameliorate (calcium-channel blockers, K(ATP)-channel openers and late sodium-channel blockers) the disease phenotype. Our study illustrates the ability of human iPSC technology to model the abnormal functional phenotype of an inherited cardiac disorder and to identify potential new therapeutic agents. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care (personalized medicine), and aid in the development of new therapies.
Nature 01/2011; 471(7337):225-9. · 36.28 Impact Factor
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ABSTRACT: Human induced pluripotent stem (hiPS) cells provide therapeutic promises, as well as a potent in vitro model for studying biological processes which take place during human embryonic development and subsequent differentiation in normal and disease states. The epigenetic characteristics of iPS cells are reprogrammed to the embryonic state at which they acquire pluripotency. In addition, telomeres in hiPS cell must elongate sufficiently to provide the necessary replicative potential. Recent studies have demonstrated that the epigenetic characteristics of telomeric and subtelomeric regions are pivotal in regulating telomere length. Here we study telomere length, subtelomeric DNA methylation and telomeric-repeat-containing RNA (TERRA) expression in several hiPS cell clones derived from normal neonatal foreskin fibroblasts. We find that telomeres lengthen significantly in hiPS cells in comparison to the parental fibroblast source, and progressively shorten after differentiation back into fibroblast-like cells, concomitantly with telomerase activation and down-regulation, respectively. Subtelomeres in hiPS cells were found to be generally hypermethylated in comparison to the parental source. However bisulfite analysis revealed that at several subtelomeres examined, methylation levels differed between hiPS clones and that both de novo methylation and demethylation processes occurred during telomere reprogramming. Notably, although subtelomeres were in general very highly methylated, TERRA levels were elevated in hiPS cells, albeit to different degrees in the various clones. TERRA elevation may reflect enhanced stability or impaired degradation in hiPS cells, and/or alternatively, increased transcription from the hypomethylated subtelomeres. We suggest that TERRA may play a role in regulation of appropriate telomere function and length in hiPS cells.
Epigenetics: official journal of the DNA Methylation Society 01/2011; 6(1):63-75. · 4.58 Impact Factor
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ABSTRACT: The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca(2+)-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).
RT-PCR and immunocytochemistry experiments identified the expression of key Ca(2+)-handling proteins. Detailed laser confocal Ca(2+) imaging demonstrated spontaneous whole-cell [Ca(2+)](i) transients. These transients required Ca(2+) influx via L-type Ca(2+) channels, as demonstrated by their elimination in the absence of extracellular Ca(2+) or by administration of the L-type Ca(2+) channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca(2+) store, contributing to [Ca(2+)](i) transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca(2+)) and ryanodine (decreasing [Ca(2+)](i)). Similarly, the importance of Ca(2+) reuptake into the SR via the SR Ca(2+) ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca(2+)](i) transients elimination. Finally, the presence of an IP3-releasable Ca(2+) pool in hiPSC-CMs and its contribution to whole-cell [Ca(2+)](i) transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phospholipase C inhibitor U73122.
Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca(2+) store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca(2+)](i) transients in hiPSC-CMs on both sarcolemmal Ca(2+) entry via L-type Ca(2+) channels and intracellular store Ca(2+) release.
PLoS ONE 01/2011; 6(4):e18037. · 4.09 Impact Factor
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ABSTRACT: Cell replacement strategies are promising interventions aiming to improve myocardial performance. Yet, the electrophysiological impact of these approaches has not been elucidated. We assessed the electrophysiological consequences of grafting of two candidate cell types, that is, skeletal myoblasts and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). The fluorescently labeled (DiO) candidate cells were grafted into the rat's left ventricular myocardium. Two weeks later, optical mapping was performed using the Langendorff-perfused rat heart preparation. Images were obtained with appropriate filters to delineate the heart's anatomy, to identify the DiO-labeled cells, and to associate this information with the voltage-mapping data (using the voltage-sensitive dye PGH-I). Histological examination revealed the lack of gap junctions between grafted skeletal myotubes and host cardiomyocytes. In contrast, positive Cx43 immunostaining was observed between donor and host cardiomyocytes in the hESC-CMs-transplanted hearts. Optical mapping demonstrated either normal conduction (four of six) or minimal conduction slowing (two of six) at the hESC-CMs engraftment sites. In contrast, marked slowing of conduction or conduction block was seen (seven of eight) at the myoblast transplantation sites. Ventricular arrhythmias could not be induced in the hESC-CM hearts following programmed electrical stimulation but were inducible in 50% of the myoblast-engrafted hearts. In summary, a unique method for assessment of the electrophysiological impact of myocardial cell therapy is presented. Our results demonstrate the ability of hESC-CMs to functionally integrate with host tissue. In contrast, transplantation of cells that do not form gap junctions (skeletal myoblats) led to localized conduction disturbances and to the generation of a proarrhythmogenic substrate.
Stem Cells 10/2010; 28(12):2151-61. · 7.78 Impact Factor
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ABSTRACT: A decade has passed since the initial derivation of human embryonic stem cells (hESC). The ensuing years have witnessed a significant progress in the development of methodologies allowing cell cultivation, differentiation, genetic manipulation, and in vivo transplantation. Specifically, the potential to derive human cardiomyocytes from the hESC lines, which can be used for several basic and applied cardiovascular research areas including in the emerging field of cardiac regenerative medicine, attracted significant attention from the scientific community. This resulted in the development of protocols for the cultivation of hESC and their successful differentiation toward the cardiomyocyte lineage fate. In this chapter, we will describe in detail methods related to the cultivation, genetic manipulation, selection, and in vivo transplantation of hESC-derived cardiomyocytes.
Methods in molecular biology (Clifton, N.J.) 01/2010; 660:85-95.
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ABSTRACT: The ability to derive human induced pluripotent stem (hiPS) cell lines by reprogramming of adult fibroblasts with a set of transcription factors offers unique opportunities for basic and translational cardiovascular research. In the present study, we aimed to characterize the cardiomyocyte differentiation potential of hiPS cells and to study the molecular, structural, and functional properties of the generated hiPS-derived cardiomyocytes.
Cardiomyocyte differentiation of the hiPS cells was induced with the embryoid body differentiation system. Gene expression studies demonstrated that the cardiomyocyte differentiation process of the hiPS cells was characterized by an initial increase in mesoderm and cardiomesoderm markers, followed by expression of cardiac-specific transcription factors and finally by cardiac-specific structural genes. Cells in the contracting embryoid bodies were stained positively for cardiac troponin-I, sarcomeric alpha-actinin, and connexin-43. Reverse-transcription polymerase chain reaction studies demonstrated the expression of cardiac-specific sarcomeric proteins and ion channels. Multielectrode array recordings established the development of a functional syncytium with stable pacemaker activity and action potential propagation. Positive and negative chronotropic responses were induced by application of isoproterenol and carbamylcholine, respectively. Administration of quinidine, E4031 (I(Kr) blocker), and chromanol 293B (I(Ks) blocker) significantly affected repolarization, as manifested by prolongation of the local field potential duration.
hiPS cells can differentiate into myocytes with cardiac-specific molecular, structural, and functional properties. These results, coupled with the potential of this technology to generate patient-specific hiPS lines, hold great promise for the development of in vitro models of cardiac genetic disorders, for drug discovery and testing, and for the emerging field of cardiovascular regenerative medicine.
Circulation 09/2009; 120(15):1513-23. · 14.74 Impact Factor
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ABSTRACT: Pro-arrhythmia (development of cardiac arrhythmias as a pharmacological side effect) has become the single most common cause of the withdrawal or restrictions of previously marketed drugs. The development of new medications, free from these side effects, is hampered by the lack of an in vitro assay for human cardiac tissue. We hypothesized that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) assessed with a combination of single cell electrophysiology and microelectrode array (MEA) mapping can serve as a novel model for electrophysiological drug screening. Current-clamp studies revealed that E-4031 and Sotalol (IKr blockers) significantly increased hESC-CM's action potential duration and also induced after-depolarizations (the in vitro correlates of increased arrhythmogenic potential). Multicellular aggregates of hESC-CMs were then analyzed with the MEA technique. Application of class I (Quinidine, Procaineamide) and class III (Sotalol) antiarrhythmic agents, E-4031, and Cisapride (a noncardiogenic agent known to lengthen QT) resulted in dose-dependent prolongation of the corrected field potential duration (cFPD). We next utilized the MEA technique to also assess pharmacological effects on conduction. Activation maps demonstrated significant conduction slowing following administration of Na channel blockers (Quinidine and Propafenone) and of the gap junction blocker (1-heptanol). While most attention has been focused on the prospects of using hESC-derived cardiomyocytes for regenerative medicine, this study highlights the possible utilization of these unique cells also for cardiac electrophysiological studies, drug screening, and target validation.
Stem cells and development 06/2008; 18(1):161-72. · 4.15 Impact Factor
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ABSTRACT: Traditional antiarrhythmic pharmacological therapies are limited by their global cardiac action, low efficacy, and significant proarrhythmic effects. We present a novel approach for the modification of the myocardial electrophysiological substrate using cell grafts genetically engineered to express specific ionic channels.
To test the aforementioned concept, we performed ex vivo, in vivo, and computer simulation studies to determine the ability of fibroblasts transfected to express the voltage-sensitive potassium channel Kv1.3 to modify the local myocardial excitable properties. Coculturing of the transfected fibroblasts with neonatal rat ventricular myocyte cultures resulted in a significant reduction (68%) in the spontaneous beating frequency of the cultures compared with baseline values and cocultures seeded with naive fibroblasts. In vivo grafting of the transfected fibroblasts in the rat ventricular myocardium significantly prolonged the local effective refractory period from an initial value of 84+/-8 ms (cycle length, 200 ms) to 154+/-13 ms (P<0.01). Margatoxin partially reversed this effect (effective refractory period, 117+/-8 ms; P<0.01). In contrast, effective refractory period did not change in nontransplanted sites (86+/-7 ms) and was only mildly increased in the animals injected with wild-type fibroblasts (73+/-5 to 88+/-4 ms; P<0.05). Similar effective refractory period prolongation also was found during slower pacing drives (cycle length, 350 to 500 ms) after transplantation of the potassium channels expressing fibroblasts (Kv1.3 and Kir2.1) in pigs. Computer modeling studies confirmed the in vivo results.
Genetically engineered cell grafts, transfected to express potassium channels, can couple with host cardiomyocytes and alter the local myocardial electrophysiological properties by reducing cardiac automaticity and prolonging refractoriness.
Circulation 03/2008; 117(6):720-31. · 14.74 Impact Factor
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ABSTRACT: We evaluated the ability of human embryonic stem cells (hESCs) and their cardiomyocyte derivatives (hESC-CMs) to engraft and improve myocardial performance in the rat chronic infarction model.
Cell therapy is emerging as a novel therapy for myocardial repair but is hampered by the lack of sources for human cardiomyocytes.
Immunosuppressed healthy and infarcted (7 to 10 days after coronary ligation) rat hearts were randomized to injection of undifferentiated hESCs, hESC-CMs, noncardiomyocyte hESC derivatives, or saline. Detailed histological analysis and sequential echocardiography were used to determine the structural and functional consequences of cell grafting.
Transplantation of undifferentiated hESCs resulted in the formation of teratoma-like structures. This phenomenon was prevented by grafting of ex vivo pre-differentiated hESC-CMs. The grafted cardiomyocytes survived, proliferated, matured, aligned, and formed gap junctions with host cardiac tissue. Functionally, animals injected with saline or nonmyocyte hESC derivatives demonstrated significant left ventricular (LV) dilatation and functional deterioration, whereas grafting of hESC-CMs attenuated this remodeling process. Hence, post-injury baseline fractional shortening deteriorated by 50% (from 20 +/- 2% to 10 +/- 2%) and by 30% (20 +/- 2% to 14 +/- 2%) in the saline and nonmyocyte groups while improving by 22% (21 +/- 2% to 25 +/- 3%) in the hESC-CM group. Similarly, wall motion score index and LV diastolic dimensions were significantly lower in the hESC-CM animals.
Transplantation of hESC-CMs after extensive myocardial infarction in rats results in the formation of stable cardiomyocyte grafts, attenuation of the remodeling process, and functional benefit. These findings highlight the potential of hESCs for myocardial cell therapy strategies.
Journal of the American College of Cardiology 12/2007; 50(19):1884-93. · 14.16 Impact Factor
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ABSTRACT: Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomyocytes. Successful application of these unique cells in the areas of cardiovascular research and regenerative medicine has been hampered by difficulties in identifying and selecting specific cardiac progenitor cells from the mixed population of differentiating cells. We report the generation of stable transgenic hESC lines, using lentiviral vectors, and single-cell clones that express a reporter gene (eGFP) under the transcriptional control of a cardiac-specific promoter (the human myosin light chain-2V promoter). Our results demonstrate the appearance of eGFP-expressing cells during the differentiation of the hESC as embryoid bodies (EBs) that can be identified and sorted using FACS (purity>95%, viability>85%). The eGFP-expressing cells were stained positively for cardiac-specific proteins (>93%), expressed cardiac-specific genes, displayed cardiac-specific action-potentials, and could form stable myocardial cell grafts following in vivo cell transplantation. The generation of these transgenic hESC lines may be used to identify and study early cardiac precursors for developmental studies, to robustly quantify the extent of cardiomyocyte differentiation, to label the cells for in vivo grafting, and to allow derivation of purified cell populations of cardiomyocytes for future myocardial cell therapy strategies.
The FASEB Journal 08/2007; 21(10):2551-63. · 5.71 Impact Factor
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ABSTRACT: Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.
Nature Biotechnology 11/2004; 22(10):1282-9. · 23.27 Impact Factor
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ABSTRACT: Human embryonic stem cell-derived cardiomyocytes (hES-CMs) are thought to recapitulate the embryonic development of heart cells. Given the exciting potential of hES-CMs as replacement tissue in diseased hearts, we investigated the pharmacological sensitivity and ionic current of mid-stage hES-CMs (20-35 days post plating). A high-resolution microelectrode array was used to assess conduction in multicellular preparations of hES-CMs in spontaneously contracting embryoid bodies (EBs). TTX (10 microm) dramatically slowed conduction velocity from 5.1 to 3.2 cm s(-1) while 100 microm TTX caused complete cessation of spontaneous electrical activity in all EBs studied. In contrast, the Ca2+channel blockers nifedipine or diltiazem (1 microm) had a negligible effect on conduction. These results suggested a prominent Na+ channel current, and therefore we patch-clamped isolated cells to record Na+ current and action potentials (APs). We found for isolated hES-CMs a prominent Na+ current (244 +/- 42 pA pF(-1) at 0 mV; n=19), and a hyperpolarization-activated current (HCN), but no inward rectifier K+ current. In cell clusters, 3 microm TTX induced longer AP interpulse intervals and 10 microm TTX caused cessation of spontaneous APs. In contrast nifedipine (Ca2+ channel block) and 2 mm Cs+ (HCN complete block) induced shorter AP interpulse intervals. In single cells, APs stimulated by current pulses had a maximum upstroke velocity (dV/dtmax) of 118 +/- 14 V s(-1) in control conditions; in contrast, partial block of Na+ current significantly reduced stimulated dV/dtmax (38 +/- 15 V s(-1)). RT-PCR revealed NaV1.5, CaV1.2, and HCN-2 expression but we could not detect Kir2.1. We conclude that hES-CMs at mid-range development express prominent Na+ current. The absence of background K+ current creates conditions for spontaneous activity that is sensitive to TTX in the same range of partial block of NaV1.5; thus, the NaV1.5 Na+ channel is important for initiating spontaneous excitability in hES-derived heart cells.
The Journal of Physiology 10/2004; 559(Pt 2):479-96. · 4.72 Impact Factor
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Methods in Enzymology 02/2003; 365:461-73. · 2.04 Impact Factor
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Methods in molecular biology (Clifton, N.J.) 02/2002; 189:199-206.
