Creation of human cardiac cell sheets using pluripotent stem cells.

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets, the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study, we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation, cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating, even after cell sheets were detached from culture dishes. Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue.

Neoechinulin a imparts resistance to acute nitrosative stress in PC12 cells: a potential link of an elevated cellular reserve capacity for pyridine nucleotide redox turnover with cytoprotection.

Treatment of PC12 cells with fungus-derived alkaloid neoechinulin A for more than 12 h renders the cells resistant to subsequent superoxide (O2⁻)/nitric oxide (NO) insults derived from 3-morpholinosydnonimine (SIN-1). However, the underlying mechanism(s) remains largely unclear. To elucidate the mechanism(s), we assessed the specificity of the cytoprotection afforded by neoechinulin A treatment using other cytocidal stressors and also clarified the resulting cellular alterations, focusing on the antioxidant and metabolic enzymes systems. Neoechinulin A treatment for more than 12 h endowed PC12 cells with significant resistance to transient NO toxicity, but not persistent NO toxicity, bolus H2O2 toxicity, or oxidative insult from the redox cycling quinone menadione. Cellular antioxidant system profiling revealed no substantial potentiation of the activity of any antioxidant enzyme in lysate from the neoechinulin A-treated cells excluding glutathione (GSH) content, which was significantly decreased (>50%), resulting in a proportional compromise in the thiol-reducing activity of the intact cells. In addition, no differences were observed in the activity for any nicotinamide adenine dinucleotide (phosphate) reduced form (NAD(P)H)-generating enzyme, steady-state NAD(P)H/nicotinamide adenine dinucleotide (phosphate) oxidized form (NAD(P)⁺) ratios, or the levels of total NAD(P)H. Nevertheless, the neoechinulin A-treated intact cells exhibited increased NAD(P)H redox turnover when driven by extracellular tetrazolium. The structurally inactive analog preechinulin failed to protect cells against NO toxicity or induce these alterations, suggesting their link with the cytoprotective mechanism. These results suggest that neoechinulin A, despite disabling the GSH defense system, confers cytoprotection against nitrosative stresses by elevating the cellular reserve capacity for NAD(P)H generation, which could offset crippling of energy-supplying systems due to nitrosative stress.

Advanced fibrosis of non-alcoholic steatohepatitis affects the significance of lipoprotein(a) as a cardiovascular risk factor.

BACKGROUND AND AIMS: Lipoprotein(a) [Lp(a)] is an important independent cardiovascular risk factor. However, Lp(a) levels are lower in patients with chronic liver disease than in healthy subjects. Furthermore, Lp(a) levels decrease as residual liver function declines. Although non-alcoholic fatty liver disease (NAFLD), especially advanced non-alcoholic steatohepatitis (NASH), increases the risk of cardiovascular diseases, the relationship between serum Lp(a) level and NASH is unknown. Thus, we examined the relationship between serum Lp(a) levels and biopsy-proved NAFLD and clarified the significance of Lp(a) measurements for cardiovascular disease screening in patients with NAFLD. METHODS: A total of 176 patients with NAFLD were enrolled. Comprehensive blood chemistry tests and histological examinations of liver samples were conducted. The relationship between serum Lp(a) levels and NAFLD was analyzed. RESULTS: Serum Lp(a) levels in advanced fibrosis (stage 3-4) were lower than those in non-advanced fibrosis (stage 0-2) (p < 0.05). After adjustment for age, sex, body mass index, alanine aminotransferase (ALT), creatinine (Cre), HbA1c level, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and the use of lipid-lowering agents, the significant inverse association between advanced fibrosis and serum Lp(a) levels remained (p < 0.01). Although the Lp(a) level was inversely associated with an NAFLD Activity Score (NAS) of 5-8, there was no significant association between Lp(a) levels and NAS adjusted for age, sex, body mass index, ALT, Cre, HbA1c level, HDL-C, LDL-C, TG, and the use of lipid-lowering agents. CONCLUSIONS: Advanced NASH is associated with low serum Lp(a) levels; therefore, Lp(a) levels may not be useful in evaluating cardiovascular risk.

SIN-1 cytotoxicity to PC12 cells is mediated by thiol-sensitive short-lived substances generated through SIN-1 decomposition in culture medium.

As a generator of peroxynitrite (ONOO(-)), 3-morpholinosydnonimine (SIN-1) is widely used in the study of oxidative/nitrosative stress in cultured cells, although controversy exists regarding active species responsible for cytotoxicity. In this study, we report that unstable thiol-sensitive substances, generated from the reaction of SIN-1 with components in culture medium, play a crucial role in SIN-1 cytotoxicity in PC12 cells. Exposure of cells to culture medium obtained after almost complete SIN-1 decomposition at 37 degrees C for 2h demonstrated almost the same degree of cytotoxicity as did fresh SIN-1. The cytotoxicity of SIN-1-decomposed medium largely depended on serum, decayed with time, and could be completely abolished by the addition of thiols. Degradation of synthetic ONOO(-) in the culture medium did not reproduce the unstable cytotoxicity. The presence of superoxide dismutase (SOD) during SIN-1 decomposition prevented the formation of the cytotoxic substances, whereas SOD had no protection against the cytotoxicity itself, suggesting a crucial role of simultaneously generated superoxide and nitric oxide in the formation of the toxicants, but not in their cytotoxic action. The cytotoxicity of fresh SIN-1 is dramatically suppressed in a basal medium (Hanks balanced salt), suggesting that the cytotoxicity of fresh SIN-1 also requires components of culture medium. These results suggest that SIN-1 cytotoxicity in PC12 cells is mediated via the generation of cytotoxic substances in the medium during its decomposition.

Anaerobic glycerol-3-phosphate dehydrogenase complex from hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Glycerol-3-phosphate (G3P) is a key intermediate of glycerol metabolism and is oxidized to dihydroxyacetone phosphate aerobically or anaerobically by appropriate G3P dehydrogenases. A hyperthermophilic archaeon Thermococcus kodakarensis KOD1 has a novel operon consisting of three genes encoding an anaerobic G3P dehydrogenase (G3PDH), an NADH oxidase (NOX), and a molybdopterin oxidoreductase (MOX). Typically, the G3PDH gene (glpA) is included in an operon with genes encoding essential subunits of the G3PDH complex, glpB and glpC. The three genes from T. kodakarensis were cloned and expressed in Escherichia coli, and their recombinant proteins, Tk-G3PDH, Tk-NOX and Tk-MOX, were characterized. The optimal temperature of Tk-G3PDH for activity was 80°C, indicating high thermal stability. Tk-G3PDH has flavin adenine dinucleotide as a prosthetic group and catalyzes oxidation of G3P with kcat/Km 1.93 × 103 M-1s-1 at 80°C, compared with 9.83 × 105 M-1s-1 for the E. coli G3PDH complex at 37°C. Interestingly, Tk-G3PDH can catalyze this reaction even as a monomer, whereas GlpA must form a complex with GlpB and GlpC. Tk-G3PDH also forms a putative heteropentamer with Tk-NOX and Tk-MOX (G3PDH:NOX:MOX = 2:2:1). This complex may form an electron transfer pathway to a final electron acceptor in the cell membrane, as is the case for the typical G3PDH complex GlpABC.

Bicarbonate plays a critical role in the generation of cytotoxicity during SIN-1 decomposition in culture medium.

3-Morpholinosydnonimine (SIN-1) is used as a donor of peroxynitrite (ONOO(-)) in various studies. We demonstrated, however, that, the cell-culture medium remains cytotoxic to PC12 cells even after almost complete SIN-1 decomposition, suggesting that reaction product(s) in the medium, rather than ONOO(-), exert cytotoxic effects. Here, we clarified that significant cytotoxicity persists after SIN-1 decomposes in bicarbonate, a component of the culture medium, but not in NaOH. Cytotoxic SIN-1-decomposed bicarbonate, which lacks both oxidizing and nitrosating activities, degrades to innocuous state over time. The extent of SIN-1 cytotoxicity, irrespective of its fresh or decomposed state, appears to depend on the total number of initial SIN-1 molecules per cell, rather than its concentration, and involves oxidative/nitrosative stress-related cell damage. These results suggest that, despite its low abundance, the bicarbonate-dependent cytotoxic substance that accumulates in the medium during SIN-1 breakdown is the cytotoxic entity of SIN-1.

Fabrication of mouse embryonic stem cell-derived layered cardiac cell sheets using a bioreactor culture system.

Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1 × 10(8) cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5 × 10(8). Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.