SummaryCardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 106 hiPSCs per layer yielded 7.2 × 108 hiPSCs in 4-layer CPs and 1.7 × 109 hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%–87%. Approximately 6.2–7.0 × 108 cells (4-layer) and 1.5–2.8 × 109 cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future.

Female sex is an independent risk factor for development of torsade de pointes (TdP) arrhythmias not only in congenital long QT syndromes but also in acquired long QT syndromes. Clinical and experimental evidences suggest that the gender differences may be due to, at least in part, gender differences in regulation of rate-corrected QT (QTC) interval between men and women. In adult women, both QTC interval and arrhythmic risks in TdP alter cyclically during menstrual cycle, suggesting a critical role of female sex hormones in cardiac repolarization process. These gender differences in fundamental cardiac electrophysiology result from variable ion channel expression and diverse sex hormonal regulation via long term genomic and acute non-genomic actions, and sex differences in drug responses and metabolisms. In particular, non-genomic actions of testosterone and progesterone on cardiac ion channels are likely to contribute to the gender differences in cardiac repolarization processes. This review summarizes current knowledge on sex hormonal regulation of cardiac ion channels which contribute to cardiac repolarization processes and its implication for gender differences in drug-induced long QT syndromes.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes hold great potentials to predict pro-arrhythmic risks in preclinical cardiac safety screening, although the hiPSC cardiomyocytes exhibit rather immature functional and structural characteristics, including spontaneous activity. Our physiological characterization and mathematical simulation showed that low expression of the inward-rectifier potassium (I) channel is a determinant of spontaneous activity. To understand impact of the low I expression on the pharmacological properties, we tested if transduction of hiPSC-derived cardiomyocytes with KCNJ2, which encodes the I channel, alters pharmacological response to cardiac repolarization processes. The transduction of KCNJ2 resulted in quiescent hiPSC-derived cardiomyocytes, which need pacing to elicit action potentials. Significant prolongation of paced action potential duration in KCNJ2-transduced hiPSC-derived cardiomyocytes was stably measured at 0.1 μM E-4031, although the same concentration of E-4031 ablated firing of non-treated hiPSC-derived cardiomyocytes. These results in single cells were confirmed by mathematical simulations. Using the hiPSC-derived cardiac sheets with KCNJ2-transduction, we also investigated effects of a range of drugs on field potential duration recorded at 1 Hz. The KCNJ2 overexpression in hiPSC-derived cardiomyocytes may contribute to evaluate a part of QT-prolonging drugs at toxicological concentrations with high accuracy.

In V(D)J joining of antigen receptor genes, two recombination signal sequences (RSSs), 12-RSS and 23-RSS, are paired and complexed with the protein products of recombination-activating genes RAG1 and RAG2. Using magnetic beads, we purified the pre-and postcleavage complexes of V(D)J joining and analyzed them by DNase I footprinting. In the precleavage synaptic complex, strong protection was seen not only in the 9-mer and spacer regions but also near the coding border of the 7-mer. This is a sharp contrast to the single RSS-RAG complex where the 9-mer plays a major role in the interaction. We also analyzed the postcleavage signal end complex by footprinting. Unlike what was seen with the precleavage complex, the entire 7-mer and its neighboring spacer regions were protected. The present study indicates that the RAG-RSS interaction in the 7-mer region drastically changes once the synaptic complex is formed for cleavage.V(D)J recombination plays key roles in the activation and diversification of the antigen receptor genes (39). Two pairs of recombination signal sequences (RSSs), 7-mer (CACAGTG) and 9-mer (ACAAAAACC), are required for this type of recombination (22,30). Furthermore, the spacer separating the 7-mer and 9-mer is either 12 or 23 bp in length. V(D)J recombination takes place between two RSSs with different spacer lengths, one containing a 12-bp spacer (12-RSS) and the other containing a 23-bp spacer (23-RSS) (7,31,32). This is the so-called 12/23-bp spacer rule for V(D)J recombination (4).In the initial step of V(D)J joining, each RSS is individually recognized by the protein products of the recombination-activating genes RAG1 and RAG2 (3,15,26). This RSS-RAG interaction primarily involves the Hin homeodomain of the RAG1 protein and the 9-mer region of RSS DNA (6,26,34,35). The next step is the formation of the synaptic complex, where the two RSS-RAG complexes are brought together (8,24,25,40) in the presence of a DNA-bending protein, HMG1 (18,33,41,42,43). When the 12/23 joining rule is satisfied, RSS DNA is then cleaved in two successive steps, nicking and hairpin formation (23). After the cleavage, the two coding ends and the two signal ends (SEs) recombine to form a coding joint and a signal joint, respectively, by DNA repair mechanisms (5,10,12,13,14,21,27,28,29,38,44).In order to study the RSS-RAG interaction during the process of V(D)J joining, we purified the pre-and postcleavage complexes by using RSS DNA and RAG proteins in vitro and analyzed them by DNase I footprinting. The present results indicate that the mode of RAG-RSS interaction in the 7-mer drastically changes once the two RSS-RAG complexes are associated to form the synaptic complex. MATERIALS AND METHODSPreparation of proteins. Truncated RAG1 and RAG2 fused with maltose binding protein were expressed in Sf.9 cells by using a baculovirus vector and were purified as previously described (40). Porcine HMG1 protein was prepared as previously described (1).DNA substrates. Oligonucleotides were chemically synthesized and were purified by ...

An 8-km long MgB2 wire for a prototype klystron magnet was made and evaluated. The wire was made by a typical in situ method; it has 10 filaments and 0.67 mm in outer diameter. The homogeneity of Ic of this wire was evaluated by several methods. Deviation of Ic values in short sample wires was very small. In addition, the current sharing temperature of the MgB2 magnet (made of two reels of wire 2.9 km long each) agreed well with the estimated value of the Ic-B-T properties in short sample wires. Based on the obtained results, it can be said that the Ic properties of the entire wire length are quite uniform.

Owing to the relatively high critical temperature and the low manufacturing cost, MgB2 superconducting wires are promising for liquid helium-free superconducting applications. Today, commercially available MgB2 wires are manufactured by either an in situ or ex situ powder-in-tube process, the in situ process being more effective to obtain high critical current density. In in situ-processed wires, however, the critical current density is seriously suppressed by the high porosity of MgB2 filaments. To resolve this problem, we propose an innovative method of using precursor powder prepared by mechanical milling of magnesium, boron, and coronene powders. This precursor powder has a metal–matrix–composite structure, in which boron particles are dispersed in a magnesium matrix. The plastic deformation of the precursor powder through wire processing leads to compact packing, and a dense MgB2 filament is generated after heat treatment. As a result, the limitation of critical current density that occurs for the typical in situ process is overcome, and the practical critical current density of 103 A mm−2 is obtained at 10 K and 6.1 T, at 15 K and 4.8 T, and at 20 K and 3.3 T.

A comparative study of in situ MgB 2 wire and MgB 2 bulk was carried out to clarify the mechanism for the high critical current density, J c , in the practical in situ MgB 2 wires. The in situ MgB 2 wire was manufactured with an area-reduction ratio of 99.93%, which was one of the highest values in MgB 2 superconducting wires previously reported. The electrical connectivity, K , and the flux pinning strength, F p , which are important factors in explaining the behavior of J c , could be determined in the same manner as those for the bulk sample; K was well understood with the three-dimensional percolation model, and F p was effectively explained by the electron scattering mechanism by grain boundaries. On the other hand, the area-reduction process dramatically enhanced the value of K , leading to an increase in the value of J c . The respective values of K and J c (20 K, ∼0 T) reached 0.24-0.34 and 6.0 × 10 3 -8.4 × 10 3 A mm −2 , which were twice or three times higher than those of typical in situ bulks. This is because the plastic deformation of magnesium particles increased the packing factor of raw powders through a repetitive drawing process.

We are now developing a new in situ deposition process for MgB 2 film as a candidate method to massproduce MgB 2 thin film superconducting tape. In the new method, a MgB 2 film is deposited on a heated metal substrate by a hybrid deposition method, which consists of thermal evaporation of magnesium and sputtering of boron. By using the hybrid deposition method, the substrate temperature can raise from 250 to 350°C, while its fluctuation is kept less than 1°C, which will improve the quality and reproducibility of MgB 2 film in mass production. The J c of MgB 2 film deposited by the hybrid deposition method at 20 K and self-field was more than 30,000 A mm −2 , which was better than the results reported by the two-step in situ process using DC sputtering and 830°C high-temperature post annealing [1] or by the as-grown depostion using sputttering targets of Mg and B [2]. Although we obtained better J c than other deposition methods that use sputtering process, the J c is still lower than the value we obtained by using a co-evaporation method with electron beam (EB). We investigated the film structure and J c -B-T properties of the film made by the hybrid deposition method and compared them with those of the film made by co-evaporation. From the analysis results, we think the reasons for the lower J c are the larger amount of heterogeneous phases such as magnesium oxides in the film and the amorphous B phase under the MgB 2 . We expect to improve the the crystal qualities and superconductivities of the MgB 2 film deposited by the new method by removing impurities in Ar gas during sputtering and thinning the B amorphous phase by increasing the Mg deposition rate in the initial stage of deposition.