SummaryHigh-purity cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are promising for drug development and myocardial regeneration. However, most hiPSC-derived CMs morphologically and functionally resemble immature rather than adult CMs, which could hamper their application. Here, we obtained high-quality cardiac tissue-like constructs (CTLCs) by cultivating hiPSC-CMs on low-thickness aligned nanofibers made of biodegradable poly(D,L-lactic-co-glycolic acid) polymer. We show that multilayered and elongated CMs could be organized at high density along aligned nanofibers in a simple one-step seeding process, resulting in upregulated cardiac biomarkers and enhanced cardiac functions. When used for drug assessment, CTLCs were much more robust than the 2D conventional control. We also demonstrated the potential of CTLCs for modeling engraftments in vitro and treating myocardial infarction in vivo. Thus, we established a handy framework for cardiac tissue engineering, which holds high potential for pharmaceutical and clinical applications.

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Introduction: Functional skeletal myoblasts (SMBs) are transplanted into the heart effectively and safely as cell sheets, which induce functional recovery in myocardial infarction (MI) patients without lethal arrhythmia. However, their therapeutic effect is limited by ischemia. Mesenchymal stem cells (MSCs) have prosurvival/ proliferation and antiapoptotic effects on co-cultured cells in vitro. We hypothesized that adding MSCs to the SMB cell sheets might enhance SMB survival post-transplantation and improve their therapeutic effects. Methods and Results: Cell sheets of primary SMBs of male Lewis rats (r-SMBs), primary MSCs of human female fat tissues (h-MSCs), and their co-cultures were generated using temperature-responsive dishes. The levels of candidate paracrine factors, rat hepatocyte growth factor and vascular endothelial growth factor, in vitro were significantly greater in the h-MSC/r-SMB co-cultures than in those containing r-SMBs only, by real-time PCR and enzyme-linked immunosorbent assay (ELISA). MI was generated by left-coronary artery occlusion in female athymic nude rats. Two weeks later, co-cultured r-SMB or h-MSC cell sheets were implanted or no treatment was performed (n = 10 each). Eight weeks later, systolic and diastolic function parameters were improved in all three treatment groups compared to no treatment, with the greatest improvement in the co-cultured cell sheet transplantation group. Consistent results were found for capillary density, collagen accumulation, myocyte hypertrophy, Akt-signaling, STAT3 signaling, and survival of transplanted cells of rat origin, and were related to poly (ADP-ribose) polymerase-dependent signal transduction. Conclusions: Adding MSCs to SMB cell sheets enhanced the sheets' angiogenesis-related paracrine mechanics and, consequently, functional recovery in a rat MI model, suggesting a possible strategy for clinical applications.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral nerve disorder. The causative gene for axonal type CMT2E has been identified as neurofilament light (NF-L) chain. Using cultured cells and in vitro assays, we analyzed the filament formation ability of Pro22 CMT mutant proteins of NF-L, P22S and P22T. NF-L Pro22 mutant proteins formed large aggregates in SW13- cells and cortical neurons and assembled into short twisty threads thinner than 10 nm filaments in vitro. Those threads associated with each other at their ends and entangled into large aggregates, also abnormalities, were detected at steps in oligomer formation. Pro22 mutations abolished Thr21 phosphorylation by cyclin-dependent kinase 5 and external signal regulated kinase, which suppressed filament assembly, but phosphorylation by protein kinase A (PKA) inhibited aggregate formation in vitro and alleviated aggregates in cortical neurons. These results indicate that the Pro22 CMT mutation induces abnormal filament aggregates by disrupting proper oligomer formation and the aggregates are mitigated by phosphorylation with PKA, which makes it a viable target for the development for therapeutics.

Cell-sheet transplantation induces angiogenesis for chronic myocardial infarction (MI), though insufficient capillary maturation and paucity of arteriogenesis may limit its therapeutic effects. Omentum has been used clinically to promote revascularization and healing of ischemic tissues. We hypothesized that cell-sheet transplantation covered with an omentum-flap would effectively establish mature blood vessels and improve coronary microcirculation physiology, enhancing the therapeutic effects of cell-sheet therapy. Rats were divided into four groups after coronary ligation; skeletal myoblast cell-sheet plus omentum-flap (combined), cell-sheet only, omentum-flap only, and sham operation. At 4 weeks after the treatment, the combined group showed attenuated cardiac hypertrophy and fibrosis, and a greater amount of functionally (CD31(+)/lectin(+)) and structurally (CD31(+)/α-SMA(+)) mature blood vessels, along with myocardial upregulation of relevant genes. Synchrotron-based microangiography revealed that the combined procedure increased vascularization in resistance arterial vessels with better dilatory responses to endothelium-dependent agents. Serial (13)N-ammonia PET showed better global coronary flow reserve in the combined group, mainly attributed to improvement in the basal left ventricle. Consequently, the combined group had sustained improvements in cardiac function parameters and better functional capacity. Cell-sheet transplantation with an omentum-flap better promoted arteriogenesis and improved coronary microcirculation physiology in ischemic myocardium, leading to potent functional recovery in the failing heart.

Intramyocardial delivery of ONO1301SR, which is a PLGA-coated slow-releasing form of ONO1301, up-regulated multiple cardiotherapeutic factors in the injected territory, leading to region-specific reverse left ventricular remodeling and consequently a global functional recovery in a rapid-pacing-induced canine DCM model, warranting a further preclinical study to optimize this novel drug-delivery system to treat DCM.

Resveratrol has been extensively investigated because of its beneficial effects in delaying age-related diseases, thus extending the lifespan, possibly by mimicking calorie restriction. For this study, cell biological techniques were used to examine how resveratrol influenced hepatocytes in a senescence-accelerated mouse P10 (SAMP10), treated from 35 to 55 weeks of age, with special emphasis on the relationship between mitochondria and lipid droplets. Survival ratio, body weight and food intake of SAMP10 did not differ significantly between the control and resveratrol-treated groups. Compared with the control, the treated livers were altered significantly, as follows. Lipid droplets were reduced and mitochondria were increased in number in hepatocytes. Phosphorylation of acetyl-CoA carboxylase and the expression of both the mitochondrial ATP synthase β subunit and Mn superoxide dismutase (SOD2) were increased. Mitochondria, expressing more SOD2, were more tightly associated with lipid droplets, suggesting the enhancement of lipolysis through the activation of mitochondrial functions. Cathepsin D expression was less in hepatocytes but enhanced in Kupffer cells, which were increased in number and size with more numerous lysosome-related profiles. Together, resveratrol may activate mitochondria resulting in consuming lipids, and may also activate Kupffer cells by which a beneficial milieu for hepatocytes may be created. Both might be related to improvement in the functioning of the liver, which is the organ that is central to metabolic regulation.

Tocopherols and tocotrienols constitute the vitamin E family. Although alpha-tocotrienol is the most neuroprotective form of vitamin E proved to be effective against stroke, alpha-tocopherol is the most abundant in nature and is used most often for disease prevention/treatment. A recent metaanalysis of human studies suggested that alpha-tocopherol supplementation increases all-cause mortality. Therefore, we investigated the effects of alpha-tocopherol ( approximately 44 mg/kg body weight; equivalent to 2,600 mg/human/day) on the central nervous system (CNS) of stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP treated with high dose alpha-tocopherol had significantly higher blood pressure than untreated controls fed a basal diet that contained approximately 4 mg tocopherols/kg body weight, but neither group experienced a change in degree of lipid peroxidation in serum or CNS tissue. Biochemical/immunohistochemical analyses demonstrated that expressions of phosphorylated neurofilament H protein, glial fibrillary acidic protein and cathepsin D in the CNS tissue were significantly enhanced in alpha-tocopherol-supplemented rats, whereas expressions of SOD2 and Bcl-xL were diminished in response to alpha-tocopherol supplementation. Similarly, the frequency of cathepsin D-positive cells, corresponding mostly to microglial cells, was significantly increased in alpha-tocopherol-supplemented rats. Alpha-tocopherol supplementation also increased the number of lysosomes and lipofuscin granules in perikarya of both hippocampal pyramidal and Purkinje cells. Furthermore, alpha-tocopherol supplementation increased the frequency of glial filaments and lipofuscin granules in astrocytes and lysosomes in microglial cells that were frequently occupied with phagocytosed inclusion structures. The present results are the first to suggest that a very high dose of alpha-tocopherol supplementation increases blood pressure in SHRSP rats and influences the CNS tissue in a manner that seems adverse.