BACKGROUND:-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. Methods and Results-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 (IKr blocker), and chromanol 293B (IKs blocker) significantly affected repolarization, as manifested by prolongation of the local field potential duration. Conclusions-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.

The L-type calcium channel (LTCC) isoforms Cav1.2 and Cav1.3 display similar 1,4-Dihydropyridine binding properties and are both expressed in mammalian brain. Recent work implicates Cav1.3 channels as interesting drug targets, but no isoform selective modulators exist. It is also unknown to which extent Cav1.1 and Cav1.4 contribute to L-type specific dihydropyridine (DHP) binding activity in brain. To address this question and to determine if DHPs can discriminate between Cav1.2 and Cav1.3 binding pockets we combined radioreceptor assays and quantitative PCR (qPCR). We bred double mutants (Cav-DM) from mice expressing mutant Cav1.2 channels (Cav1.2DHP(-/-)) lacking high affinity for DHPs and from Cav1.3 knockouts (Cav1.3(-/-)).(+)-[(3)H]isradipine binding to Cav1.2DHP(-/-) and Cav-DM brains was reduced to 15.1 and 4.4% of wildtype, respectively, indicating that Cav1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Cav1.1 and Cav1.4 alpha1 subunits comprised 0.08% of the LTCC transcripts in mouse whole brain, suggesting that they cannot account for the residual binding. Instead, this could be explained by low affinity binding (127-fold Kd increase) to the mutated Cav1.2 channels. Inhibition of (+)-[(3)H]isradipine binding to Cav1.2DHP(-/-)(predominantly Cav1.3) and wildtype (predominantly Cav1.2) brain membranes by unlabeled DHPs revealed a 3-4-fold selectivity of nitrendipine and nifedipine for the Cav1.2 binding pocket, a finding further confirmed with heterologously expressed channels. This suggests that small differences in their binding pockets may allow development of isoform-selective modulators for LTCCs and that, due to their very low expression, Cav1.1 and Cav1.4 are unlikely to serve as drug targets to treat CNS diseases.

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) anti-arrhythmic agents, E4031, and Cisapride (a non-cardiogenic 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). Conclusions: 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.

In recent years, clusters of Pneumocystis jirovecii (formerly P. carinii) pneumonia (PCP) among immunocompromised individuals were reported. Mostly, the source of infections was suspected within the clinical settings when transplant recipients and PCP patients shared hospital facilities. We report on a cluster of 16 renal transplant recipients positive for P. jirovecii. None of them received anti-Pneumocystis prophylaxis prior to P. jirovecii detection. Epidemiological studies revealed that 15 of them were kidney transplanted at a German university hospital and attended the same inpatient and outpatient clinic from January through September, 2006. Multilocus sequence typing (MLST) was performed on the following genes: ITS1, beta-tub, 26S and mt26S. P. jirovecii DNA was available from 14 patients and showed identical MLST types among these renal transplant recipients. Surprisingly, one patient who was treated at a different nephrological centre and negated personal contacts to patients from the renal transplantation cluster harbored an identical P. jirovecii MLST type. Three HIV-positive patients and one bone-marrow transplanted hematologic malignancy patient - treated at different medical centres - were used as controls and revealed different MLST types. Interestingly, in three of the four previously described regions new alleles were detected and one new polymorphism was observed in the mt26S region. The epidemiological data and the genotyping results strongly suggest a nosocomial patient-to-patient transmission of P. jirovecii as the predominant transmission route. Therefore, a strict segregation and isolation of P. jirovecii positive/suspected patients in clinical settings seems warranted.

BACKGROUND:-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. Methods and Results-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. Conclusions-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.

OBJECTIVES: 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. BACKGROUND: Cell therapy is emerging as a novel therapy for myocardial repair but is hampered by the lack of sources for human cardiomyocytes. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A collaborative study including 13 German laboratories was conducted to evaluate the performance of two non-patented real-time PCR methods for the detection of Salmonella in milk powder targeting the ttrC/ttrA- or the invA gene. The enrichment procedure and sample DNA preparation method prior to the real-time PCR was the same for both systems and the identical DNA extraction samples were analysed. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in each laboratory as the reference. The participants received twelve coded milk powder samples each of 25 g for the analysis. Four of them were Salmonella negative (level L0), four artificially contaminated with <3 MPN/g Salmonella Typhimurium (level L1) and four artificially contaminated with 3.6 MPN/g S. Typhimurium (level L2) to the beginning of the experiment. Of the 13 laboratories 12 used various models of real-time PCR blockcyclers conducting both real-time PCR assays and three laboratories the Light Cycler 2.0 system (Roche Bioscience) conducting the ttr-based real-time PCR assay only. The relative accuracy for both real-time PCR assays performed on blockcyclers was for level L0 97.5%. For level L1 the relative accuracy was 94.1% and for level L2 it was 100% for both assays. The relative accuracy on the Light Cycler 2.0 system was 100% for all levels applied to the ttr-real-time PCR.

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.-- Huber, I., Itzhaki, I., Caspi, O., Arbel, G., Tzukerman, M., Gepstein, A., Habib, M., Yankelson, L., Kehat, I., Gepstein, L. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation.

Background: Fuchs heterochromic cyclitis (FHC) is a chronic inflammatory eye disease, usually presenting as unilateral anterior uveitis. Up to date no disease susceptibility genes have been described for FHC. Methods: The allele frequency of HLA DRB1 and DQB1, polymorphisms of the tumour necrosis factor (TNF) alpha promoter region (-376,-308,-238), the promoter (-318), first exon (+49) and (AT)n repeat polymorphism of the cytotoxic T cell antigen 4 (CTLA4) gene were analysed in 44 FHC patients and 139 healthy controls. Results: The CTLA4 -318 C/T genotype was increased in FHC patients [odds ratio (OR) 3.0, 95% confidence interval (CI) 1.4-6.5], as well as long CTLA4 (AT)n microsatellite alleles with more than 16 AT repeats (OR 2.6, 95% CI 1.3-5.3). A trend towards the -308 G/A TNF-alpha genotype was found in the patient cohort, whereas no difference in HLA class II allele distribution was observed. Conclusion:CTLA4 but not TNF-alpha or HLA class II DRB1 and DQB1 may represent a candidate gene for disease susceptibility in FHC. Copyright (c) 2007 S. Karger AG, Basel.