Noriko Matsushita

Direct comparison of different stem cell types and subpopulations reveals superior paracrine potency and myocardial repair efficacy with cardiosphere-derived cells

OBJECTIVES The goal of this study was to conduct a direct head-to-head comparison of different stem cell types in vitro for various assays of potency and in vivo for functional myocardial repair in the same mouse model of myocardial infarction. BACKGROUND Adult stem cells of diverse origins (e.g., bone marrow, fat, heart) and antigenic identity have been studied for repair of the damaged heart, but the relative utility of the various cell types remains unclear. METHODS Human cardiosphere-derived cells (CDCs), bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and bone marrow mononuclear cells were compared. RESULTS CDCs revealed a distinctive phenotype with uniform expression of CD105, partial expression of c-kit and CD90, and negligible expression of hematopoietic markers. In vitro, CDCs showed the greatest myogenic differentiation potency, highest angiogenic potential, and relatively high production of various angiogenic and antiapoptotic-secreted factors. In vivo, injection of CDCs into the infarcted mouse hearts resulted in superior improvement of cardiac function, the highest cell engraftment and myogenic differentiation rates, and the least-abnormal heart morphology 3 weeks after treatment. CDC-treated hearts also exhibited the lowest number of apoptotic cells. The c-kit(+) subpopulation purified from CDCs produced lower levels of paracrine factors and inferior functional benefit when compared with unsorted CDCs. To validate the comparison of cells from various human donors, selected results were confirmed in cells of different types derived from individual rats. CONCLUSIONS CDCs exhibited a balanced profile of paracrine factor production and, among various comparator cell types/subpopulations, provided the greatest functional benefit in experimental myocardial infarction.

Cardiospheres Recapitulate a Niche-Like Microenvironment Rich in Stemness and Cell-Matrix Interactions, Rationalizing Their Enhanced Functional Potency for Myocardial Repair†‡§

Cardiac stem cells (CSCs) are promising candidates for use in myocardial regenerative therapy. We test the hypothesis that growing cardiac‐derived cells as three‐dimensional cardiospheres may recapitulate a stem cell niche‐like microenvironment, favoring cell survival and enhancing functional benefit after transplantation into the injured heart. CSCs and supporting cells from human endomyocardial biopsies were grown as cardiospheres and compared with cells cultured under traditional monolayer condition or dissociated from cardiospheres. Cardiospheres self‐assembled into stem cell niche‐like structures in vitro in suspension culture, while exhibiting greater proportions of c‐kit+ cells and upregulated expression of SOX2 and Nanog. Pathway‐focused polymerase chain reaction (PCR) array, quantitative real‐time PCR, and immunostaining revealed enhanced expression of stem cell‐relevant factors and adhesion/extracellular‐matrix molecules (ECM) in cardiospheres including IGF‐1, histone deacetylase 2 (HDAC2), Tert, integrin‐α2, laminin‐β1, and matrix metalloproteinases (MMPs). Implantation of cardiospheres in severe combined immunodeficiency (SCID) mouse hearts with acute infarction disproportionately improved cell engraftment and myocardial function, relative to monolayer‐cultured cells. Dissociation of cardiospheres into single cells decreased the expression of ECM and adhesion molecules and undermined resistance to oxidative stress, negating the improved cell engraftment and functional benefit in vivo. Growth of cardiac‐derived cells as cardiospheres mimics stem cell niche properties with enhanced “stemness” and expression of ECM and adhesion molecules. These changes underlie an increase in cell survival and more potent augmentation of global function following implantation into the infarcted heart. STEM CELLS 2010;28:2088–2098

Expansion of human cardiac stem cells in physiological oxygen improves cell production efficiency and potency for myocardial repair.

AIMS the ex vivo expansion of cardiac stem cells from minimally invasive human heart biopsies yields tens of millions of cells within 3-4 weeks, but chromosomal abnormalities were frequently detected in preliminary production runs. Here we attempt to avoid aneuploidy and improve cell quality by expanding human cardiac stem cells in physiological low-oxygen (5% O(2)) conditions, rather than in traditional culture in a general CO(2) incubator (20% O(2)). METHODS AND RESULTS human heart biopsies (n = 16) were divided and processed in parallel to expand cardiac stem cells under 5% or 20% O(2). Compared with 20% O(2), 5% O(2) culture doubled the cell production and markedly diminished the frequency of aneuploidy. Cells expanded in 5% O(2) showed lower intracellular levels of reactive oxygen species, less cell senescence, and higher resistance to oxidative stress than those grown in 20% O(2), although the expression of stem cell antigens and adhesion molecules was comparable between groups, as was the paracrine secretion of growth factors into conditioned media. In vivo, the implantation of 5% O(2) cells into infarcted hearts of mice resulted in greater cell engraftment and better functional recovery than with conventionally cultured cells. CONCLUSION the expansion of human adult cardiac stem cells in low oxygen increased cell yield, and the resulting cells were superior by various key in vitro and in vivo metrics of cell quality. Physiological oxygen tensions in culture facilitate the ex vivo expansion of healthy, biologically potent stem cells.

Magnetic enhancement of cell retention, engraftment, and functional benefit after intracoronary delivery of cardiac-derived stem cells in a rat model of ischemia/reperfusion.

The efficiency of stem cell transplantation is limited by low cell retention. Intracoronary (IC) delivery is convenient and widely used but exhibits particularly low cell retention rates. We sought to improve IC cell retention by magnetic targeting. Rat cardiosphere-derived cells labeled with iron microspheres were injected into the left ventricular cavity of syngeneic rats during brief aortic clamping. Placement of a 1.3 Tesla magnet ~1 cm above the heart during and after cell injection enhanced cell retention at 24 h by 5.2–6.4-fold when 1, 3, or 5 × 105 cells were infused, without elevation of serum troponin I (sTnI) levels. Higher cell doses (1 or 2 × 106 cells) did raise sTnI levels, due to microvascular obstruction; in this range, magnetic enhancement did not improve cell retention. To assess efficacy, 5 × 105 iron-labeled, GFP-expressing cells were infused into rat hearts after 45 min ischemia/20 min reperfusion of the left anterior coronary artery, with and without a superimposed magnet. By quantitative PCR and optical imaging, magnetic targeting increased cardiac retention of transplanted cells at 24 h, and decreased migration into the lungs. The enhanced cell engraftment persisted for at least 3 weeks, at which time left ventricular remodeling was attenuated, and therapeutic benefit (ejection fraction) was higher, in the magnetic targeting group. Histology revealed more GFP+ cardiomyocytes, Ki67+ cardiomyocytes and GFP-/ckit+ cells, and fewer TUNEL+ cells, in hearts from the magnetic targeting group. In a rat model of ischemia/reperfusion injury, magnetically enhanced intracoronary cell delivery is safe and improves cell therapy outcomes.

Targeted MicroRNA Interference Promotes Postnatal Cardiac Cell Cycle Re-Entry.

Mammalian heart cells undergo a marked reduction in proliferative activity shortly after birth, and thereafter grow predominantly by hypertrophy. Our understanding of the molecular mechanisms underlying cardiac maturation and senescence is based largely on studies at the whole-heart level. Here, we investigate the molecular basis of the acquired quiescence of purified neonatal and adult cardiomyocytes, and use microRNA interference as a novel strategy to promote cardiomyocyte cell cycle re-entry. Expression of cyclins and cyclin-dependent kinases (CDKs) and positive modulators were down-regulated, while CDK inhibitors and negative cell cycle modulators were up-regulated during postnatal maturation of cardiomyocytes. The expression pattern of microRNAs also changed dramatically, including increases in miR-29a, miR-30a and miR-141. Treatment of neonatal cardiomyocytes with miRNA inhibitors anti-miR-29a, anti-miR-30a, and antimiR-141 resulted in more cycling cells and enhanced expression of Cyclin A2 (CCNA2). Thus, targeted microRNA interference can reactivate postnatal cardiomyocyte proliferation.

Automatic switching between the AAI and the DDD algorithm can prevent repetitive non-reentrant ventriculoatrial synchrony

A 67‐year‐old man with non‐obstructive hypertrophic cardiomyopathy had received an implantable cardioverter‐defibrillator (ICD) for an unstable, sustained ventricular tachycardia (VT) induced by programmed stimulation during an electrophysiological study 5 years earlier. An intracardiac electrogram recorded by the ICD revealed repetitive, non‐reentrant ventriculoatrial synchrony (RNRVAS) associated with hypotension. Electrophysiologic and hemodynamic studies indicated that RNRVAS was induced and reproducibly termed by a single ventricular extrastimulus from the right ventricular apex. Following attainment of the elective replacement indicator, we replaced the ICD with another having managed ventricular pacing, which automatically switched AAI and DDD, thereby avoiding unnecessary ventricular pacing. Thus far, the patient has not experienced further RNRVAS. Thus, we believe that automatic switching between AAI and DDD can prevent RNRVAS.

Case ReportEpicardial access and ventricular tachycardia ablation in a postmyocarditis patient using a nonfluoroscopic catheter visualization system

Introduction The efficacy of catheter ablation for scar-related ventricular tachycardia (VT) has significantly improved since the introduction of percutaneous epicardial access by Sosa in 1996. Percutaneous epicardial ablation has been used in approximately 30% of VT ablation cases associated with nonischemic cardiomyopathy. However, complications such as cardiac tamponade, coronary artery laceration, or occlusion develop in approximately 4% of the procedures. Epicardial puncture and ablation are indicated after coronary angiogram (CAG) so as to avoid damage to the coronary arteries. Prior to the ablation, a CAG must be repeated, and fluoroscopy is often used during the puncture while the operator’s hands are in the field. As a result, these procedures increase fluoroscopy time and radiation exposure for both the patient and the operator. MediGuide (St. Jude Medical [SJM], St. Paul, MN) is a sensor-based, electromagnetic, nonfluoroscopic catheter visualization system. The system tracks dedicated sensorembedded catheters and guidewires directly on fluoroscopy or cine-loop images acquired at the beginning of the ablation procedure. To reduce radiation exposure during the access and mapping, the MediGuide system has been used for epicardial access and ablation in our institution. In this report, we present a patient with postmyocarditis scar-related VT who underwent an ablation procedure using this technology.

High-risk transseptal puncture in a patient with a “pancake” deformity in the left atrium caused by descending aorta displacement

Catheter ablation via the transseptal approach has recently become a widely performed technique for treating atrial fibrillation (AF). However, fluoroscopic imaging provides limited anatomic guidance for the left atrial structure. We describe the case of a 78‐year‐old man who was referred to our hospital for pulmonary vein isolation for symptomatic paroxysmal AF. He had a history of pulmonary tuberculosis for which he had undergone a right upper lobectomy. A “pancake” deformity of the left atrium (LA) was observed using 64‐slice multislice computed tomography. We performed a transseptal puncture by using real‐time three‐dimensional transesophageal echocardiography (RT3D‐TEE) in combination with fluoroscopic imaging, without any complications. Although transseptal puncture can be performed without echocardiographic guidance in most patients, in our patient, RT3D‐TEE proved to be a very helpful imaging technique to access the LA.

Late Gadolinium Enhancement and Signal Average Electrocardiogram for Predicting Ventricular Tachyarrhythmia

Late gadolinium enhancement (LGE) during cardiac magnetic resonance (CMR) is associated with the prevalence of ventricular tachycardia/ventricular fibrillation (VT/VF). Signal average electrocardiogram (SAECG) is also relevant to VT/VF. The purpose of this study was to investigate the clinical significance of LGE and SAECG for patients with organic heart disease. We studied 150 patients with organic heart disease who performed CMR from 2006 to 2011. We examined them about LGE and SAECG for incidence of VT/VF. Their mean age was 62.9 years old, their mean left ventricular ejection fraction was 45.3%, and mean brain natriuretic peptide was 440 pg/ml. Their underlying heart disease were ischemic heart disease (IHD, 46 patients), dilated cardiomyopathy (38 patients), hypertrophic cardiomyopathy (HCM, 38 patients), and others. Ninety-four patients showed LGE in CMR, and 26 patients experienced VT/VF during follow-up of 13.4±9.4 months. In IHD patients, incidence of VT/VF was higher in patients with positive SAECG than patients with negative SAECG (37% vs 9%, p<0.05). In HCM patients, VT/VF occurred more frequently in patients with LGE than patients without LGE (24% vs 0%, p<0.05). In IHD, SAECG is more valuable in predicting tachyarrhythmia event. On the other hand, in HCM, LGE offers more useful information.