Electrical pulse stimulation-induced tetanic exercise simulation increases the secretion of extracellular vesicles from C2C12 myotubes.

Extracellular vesicles (EVs) released into the blood during exercise mediate its whole-body health effects. The differentiation of EVs released by skeletal muscle cells in vivo from those released by other cells is challenging, therefore, it is unclear whether exercise increases the number of EVs secreted by skeletal muscle cells. In this study, we investigated whether exercise affects the quantity of EVs released from skeletal muscle cells using in vitro exercise models. C2C12 myotubes were cultured on a gel layer with 1 or 30 Hz electrical pulse stimulation (EPS) to induce contractions as an artificial simulating exercise. We found that tetanic contraction induced by 30 Hz EPS increased the number of secreted EVs. MicroRNA (miRNA)-seq analysis revealed that 30 Hz EPS altered the miRNA in the secreted EVs. Furthermore, expression analysis of genes related to the biogenesis and transport of EVs revealed that the expression of ALG-2 interacting protein X (Alix) was increased in response to 30 Hz EPS, and the peak value of intracellular Ca2+ in myotubes at 30 Hz EPS was higher than that at 1 Hz, indicating that the increase in intracellular Ca2+ concentration may be related to the increased secretion of EVs in response to 30 Hz EPS.

Screening of anti-atrophic peptides by using photo-cleavable peptide array and 96-well scale contractile human skeletal muscle atrophy models.

Skeletal muscle atrophy is characterized by decreases in protein content, myofiber diameter, and contractile force generation. As muscle atrophy worsens the quality of life, the development of anti-atrophic substances is desirable. In this study, we aimed to demonstrate a screening process for anti-atrophic peptides using photo-cleavable peptide array technology and human contractile atrophic muscle models. We developed a 96-well system and established a screening process with less variability. Dexamethasone-induced human atrophic tissue was constructed in the system. Eight peptides were selected from the literature and used for the screening of peptides for preventing the decrease of the contractile forces of tissues. The peptide QIGFIW, which showed preventive activity, was selected as the seed sequence. As a result of amino acid substitution, we obtained QIGFIQ as a peptide with higher anti-atrophic activity. These results indicate that the combinatorial use of the photo-cleavable peptide array technology and 96-well screening system could comprise a powerful approach to obtaining anti-atrophic peptides, and suggest that the 96-well screening system and atrophic model represent a practical and powerful tool for the development of drugs/functional food ingredients.

Analysis of plasmodesmata permeability using cultured tobacco BY-2 cells entrapped in microfluidic chips.

Plasmodesmata are unique channel structures in plants that link the fluid cytoplasm between adjacent cells. Plants have evolved these microchannels to allow trafficking of nutritious substances as well as regulatory factors for intercellular communication. However, tracking the behavior of plasmodesmata in real time is difficult because they are located inside tissues. Hence, a system was constructed to monitor the movement of substances by plasmodesmata using tobacco BY-2 cells, which are linearly organized cells, and a microfluidic device that traps them in place and facilitates observation. After targeting one cell for photobleaching, recovery of the lost H2B-GFP protein was detected within 200 min. No recovery was detected in that time frame by photobleaching the entire cell filaments. This suggested that the recovery of H2B-GFP protein was not due to de novo protein synthesis, but rather to translocation from neighboring cells. The transport of H2B-GFP protein was not observed when sodium chloride, a compound known to cause plasmodesmata closure, was present in the microfluid channel. Thus, using the microfluidic device and BY-2 cells, it was confirmed that the behavior of plasmodesmata could be observed in real time under controllable conditions.

Screening of a novel free fatty acid receptor 1 (FFAR1) agonist peptide by phage display and machine learning based-amino acid substitution.

Free fatty acid receptor 1 (FFAR1 or GPR40) has attracted attention for the treatment of type 2 diabetes mellitus, and various small-molecule agonists have been developed. However, most FFAR1 agonists as well as endogenous ligands, such as linoleic acids, have high lipophilicity, and their high lipophilicity is related to off-target toxicity. Therefore, we need to focus on new ligand candidates with less toxicity. In this study, we screened peptides with FFAR1 agonist activity as new ligand candidates. First, we used phage display to identify peptides with high affinity to FFAR1. Next, the agonist activities of peptides determined by the phage display were evaluated by the TGF-α shedding assay. Finally, to improve the FFAR1 agonist activity of the peptide, we performed an inclusive single amino acid substitution and sequence analysis. Logistic regression (LR) analysis using 120 physiochemical properties was performed to predict peptides with high FFAR1 agonist activity. STTGTQY determined by phage display promoted glucose-stimulated insulin secretion in pancreatic MIN6 cells. Furthermore, STKGTF predicted by the LR analysis showed high insulin secretion at low concentrations compared to STTGTQY. The results of this study suggest that peptides could be new candidates as FFAR1 agonists.

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character.

Cyclic peptides are an attractive modality for the development of therapeutics and the identification of functional cyclic peptides that contribute to novel drug development. The peptide array is one of the optimization methods for peptide sequences and also useful to understand sequence-function relationship of peptides. Cell adherent cyclic NGR peptide which selectively binds to the aminopeptidase N (APN or CD13) is known as an attractive tumor marker. In this study, we designed and screened a library of different length and an amino acid substitution library to identify stronger cell adhesion peptides and to reveal that the factor of higher binding between CD13 and optimized cyclic peptides. Additionally, we designed and evaluated 192 peptide libraries using eight representative amino acids to reduce the size of the library. Through these optimization steps of cyclic peptides, we identified 23 peptides that showed significantly higher cell adhesion activity than cKCNGRC, which was previously reported as a cell adhesion cyclic peptide. Among them, cCRHNGRARC showed the highest activity, that is, 1.65 times higher activity than cKCNGRC. An analysis of sequence and functional data showed that the rules which show higher cell adhesion activity for the three basic cyclic peptides (cCX1 HNGRHX2 C, cCX1 HNGRAX2 C, and cCX1 ANGRHX2 C) are related with the position of His residues and cationic amino acids.

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply.

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions.

OBJECTIVES: To develop a simple pectin-degrading microorganism screening method. RESULTS: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. CONCLUSIONS: The developed method is useful for identifying pectin-degrading microorganisms.

Incorporation of Gelatin Microspheres into HepG2 Human Hepatocyte Spheroids for Functional Improvement through Improved Oxygen Supply to Spheroid Core.

The multicellular spheroid three-dimensional cell culture system can be used as a formulation for cell-based therapy. However, the viability and functions of the cells in the core region of the spheroid tend to decrease because of limited oxygen supply. In this study, we incorporated gelatin microspheres (GMS) into HepG2 human hepatocyte spheroids to allow oxygen to reach the spheroid core. GMS with an approximate diameter of 37 µm were fabricated by water-in-oil emulsification followed by freeze drying. GMS-containing HepG2 spheroids (GMS/HepG2 spheroids) were prepared by incubation of the cells with GMS at various mixing ratios in agarose gel-based microwells. Increasing the GMS ratio increased the diameter of the spheroids, and few spheroids formed with excess GMS. HepG2 cells in the GMS/HepG2 spheroids were more oxygenated than those in the GMS-free spheroids. GMS incorporation increased the viability of HepG2 cells in the spheroids and increased the CYP1A1 activity of the cells to metabolize 7-ethoxyresorufin, although mRNA expression of the CYP1A1 gene was hardly affected by GMS incorporation. These results indicate that incorporating GMS into HepG2 spheroids improves the hypoxic microenvironment in the spheroids and increases cell viability and CYP1A1 metabolic activity.

Determining Transgene Expression Characteristics Using a Suction Device with Multiple Hole Adjusting a Left Lateral Lobe of the Mouse Liver.

We developed a tissue suction-mediated transfection method (suction method) as a relatively reliable and less invasive technique for in vivo transfection. In this study, we determined hepatic transgene expression characteristics in the mouse liver, using a suction device, collecting information relevant to gene therapy and gene functional analysis by the liver suction method. To achieve high transgene expression levels, we developed a suction device with four holes (multiple hole device) and applied it to the larger portion of the left lateral lobe of the mouse liver. Hepatic transfection with physical stimuli was potentially controlled by activator protein-1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We examined the spatial distribution of transgene expression in the suctioned lobe by 2-dimensional imaging with histochemical staining and 3-dimensional multicolor deep imaging with tissue clearing methods. Through monitoring spatial distribution of transgene expression, the liver suction method was used to efficiently transfect extravascular hepatocytes in the suction-deformable upper lobe of the liver. Moreover, long-term transgene expression, at least 14 d, was achieved with the liver suction method when cytosine-phosphate-guanine (CpG)-free plasmid DNA was applied.

Alcohol-tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single-cell mutant screening system.

Enhancement of alcohol tolerance in microorganisms is an important strategy for improving bioalcohol productivity. Although cyanobacteria can be used as a promising biocatalyst to produce various alcohols directly from CO2 , low productivity, and low tolerance against alcohols are the main issues to be resolved. Nevertheless, to date, a mutant with increasing alcohol tolerance has rarely been reported. In this study, we attempted to select isopropanol (IPA)-tolerant mutants of Synechococcus elongatus PCC 7942 using UV-C-induced random mutagenesis, followed by enrichment of the tolerant candidates in medium containing 10 g/L IPA and screening of the cells with a high growth rate in the single cell culture system in liquid medium containing 10 g/L IPA. We successfully acquired the most tolerant strain, SY1043, which maintains the ability to grow in medium containing 30 g/L IPA. The photosynthetic oxygen-evolving activities of SY1043 were almost same in cells after 72 h incubation under light with or without 10 g/L IPA, while the activity of the wild-type was remarkably decreased after the incubation with IPA. SY1043 also showed higher tolerance to ethanol, 1-butanol, isobutanol, and 1-pentanol than the wild type. These results suggest that SY1043 would be a promising candidate to improve alcohol production using cyanobacteria. Biotechnol. Bioeng. 2017;114: 1771-1778. © 2017 Wiley Periodicals, Inc.

Optimization of Albumin Secretion and Metabolic Activity of Cytochrome P450 1A1 of Human Hepatoblastoma HepG2 Cells in Multicellular Spheroids by Controlling Spheroid Size.

Multicellular spheroids are useful as three-dimensional cell culture systems and for cell-based therapies. Their successful application requires an understanding of the consequences of spheroid size for cellular functions. In the present study, we prepared multicellular spheroids of different sizes using the human hepatoblastoma HepG2 cells, as hepatocytes are frequently used for in vitro drug screening and cell-based therapy. Precise polydimethylsiloxane-based microwells with widths of 360, 450, 560, and 770 µm were fabricated using a micromolding technique. Incubation of HepG2 cells in cell culture plates containing the microwells resulted in the formation of HepG2 spheroids with average diameters of 195, 320, 493, and 548 µm. The cell number per spheroid positively correlated with its diameter, and the viability of HepG2 cells was 94% or above for all samples. The smallest HepG2 spheroids showed the highest albumin secretion. On the other hand, the metabolic activity of 7-ethoxyresorufin, a fluorometric substrate for CYP1A1, increased with increasing spheroid size. These results indicate that controlling spheroid size is important when preparing HepG2 spheroids and that the size of HepG2 spheroids greatly influences the cellular function of HepG2 cells in the spheroids.

A single cell culture system using lectin-conjugated magnetite nanoparticles and magnetic force to screen mutant cyanobacteria.

Cyanobacteria can be utilized as a potential biocatalyst for the production of biofuels and biochemicals directly from CO2. Useful mutants of cyanobacteria, which can grow rapidly or are resistant to specific metabolic products, are essential to improve the productivity of biofuels. In this study, we developed a single cell culture system to effectively screen mutant cyanobacteria using magnetite nanoparticles and magnetic force. Lens culinaris Agglutinin (LCA) was selected as a lectin, which binds to the surface of Synechococcus elongatus PCC7942 cells and the LCA-conjugated magnetite cationic liposomes (MCLs) were developed for magnetic labeling of PCC7942 cells. The MCL-labeled PCC7942 cells were magnetically patterned at a single cell level by using 6,400 iron pillars of the pin-holder device. The device enabled 1,600 single cells to be arrayed in one square centimeter. We cultured the patterned cells in liquid medium and achieved higher colony-forming ratio (78.4%) than that obtained using conventional solid culture method (4.8%). Single cells with different properties could be distinguished in the single cell culture system depending on their growth. Furthermore, we could selectively pick up the target cells and subsequently perform efficient isolation culture. The ratio of successful isolation culture using the developed method was 13 times higher than that of the conventional methods. Thus, the developed system would serve as a powerful tool for screening mutant cyanobacteria.

Increased Insulin Secretion from Insulin-Secreting Cells by Construction of Mixed Multicellular Spheroids.

PURPOSE: We previously have shown that multicellular spheroids containing insulin-secreting cells are an effective therapy for diabetic mice. Here we attempted to increase insulin secretion by incorporating other cell types into spheroids. MATERIALS AND METHODS: Multicellular spheroids of mouse MIN6 pancreatic ß cells were formed in microwells alone and with aortic vascular endothelial MAEC cells or embryo fibroblast NIH3T3 cells. mRNA expression of insulin genes and insulin secretion of MIN6 cells in each spheroid were measured by real-time PCR and an insulin ELIZA kit. Moreover, collagen IV expression in each spheroid was analyzed by western blot. RESULTS: In all cases, uniformly sized (about 300 µm) multicellular spheroids were obtained. MAEC or NIH3T3 cell incorporation into MIN6 spheroids significantly increased mRNA expression of insulin genes and insulin secretion. In addition, collagen IV expression, which was reported to enhance insulin secretion from pancreatic ß cells, also increased in their spheroids. CONCLUSIONS: The formation of mixed multicellular spheroids containing collagen IV-expressing cells can improve the insulin secretion from insulin-secreting MIN6 cells, and mixed multicellular spheroids can be a potent therapeutic option for patients with type I diabetes mellitus.

Efficient capturing of circulating tumor cells using a magnetic capture column and a size-selective filter.

Detecting and analyzing circulating tumor cells (CTCs) in the blood of cancer patients is a promising approach for the early diagnosis of metastasis. Previously, we developed a size-selective filter for capturing CTCs, but its use was time consuming, particularly for capturing CTCs from large volumes of blood. In the present study, we describe the use of a magnetic capture column for rapid and efficient isolation of CTCs, which were magnetically labeled with magnetite cationic liposomes. In the capturing process, large volumes of blood containing magnetically labeled cancer cells were introduced into the column at a high flow rate to capture the cells, which were then added into the filter at a low flow rate. Our results show that the combined use of the column and filter decreased the required time for the spiked cancer cell capture, and the recovery rate of the spiked cancer cells from blood was significantly higher using the combination process (80.7 %) than that using the filter alone (64.7 %). Moreover, almost twice the number of CTCs could be captured from the blood of metastatic model mice using the combination process. These results suggest that the developed process would be useful for the rapid and efficient isolation of CTCs.

Liver suction-mediated transfection in mice using a pressure-controlled computer system.

We previously developed an in vivo tissue suction-mediated transfection method (denoted as the tissue suction method) for naked nucleic acids, such as plasmid DNA (pDNA) and small interfering RNA (siRNA), in mice. However, it remains unclear whether the suction pressure conditions affect the results of this method. Therefore, in the present study, we assembled a computer system to control the suction pressure and investigate the effects of the suction pressure conditions on the efficiency of the liver suction transfection of naked pDNA that encodes luciferase in mice. Using the developed system, we examined the effects of the minimum magnitude of the suction pressure, suction pressure waveform, and suction times of the luciferase expression level in mice livers. We determined that the liver suction method at 5 kPa was not only effective but also caused the lowest hepatic toxicity in mice. Additionally, the results indicated that the suction pressure waveform affects the luciferase expression levels, and a single period of suction on the targeted portion of the liver is sufficient for transfection. Thus, the developed system is useful for performing the tissue suction method with high accuracy and safety.

Bone morphogenetic protein signaling inhibitor improves differentiation and function of 3D muscle construct fabricated using C2C12.

Functional tissue-engineered artificial skeletal muscle tissue has great potential for pharmacological and academic applications. This study demonstrates an in vitro tissue engineering system to construct functional artificial skeletal muscle tissues using self-organization and signal inhibitors. To induce efficient self-organization, we optimized the substrate stiffness and extracellular matrix (ECM) coatings. We modified the tissue morphology to be ring-shaped under optimized self-organization conditions. A bone morphogenetic protein (BMP) inhibitor was added to improve overall myogenic differentiation. This supplementation enhanced the myogenic differentiation ratio and myotube hypertrophy in two-dimensional cell cultures. Finally, we found that myotube hypertrophy was enhanced by a combination of self-organization with ring-shaped tissue and a BMP inhibitor. BMP inhibitor treatment significantly improved myogenic marker expression and contractile force generation in the self-organized tissue. These observations indicated that this procedure may provide a novel and functional artificial skeletal muscle for pharmacological studies.

Metabolic flux analysis of genetically engineered Saccharomyces cerevisiae that produces lactate under micro-aerobic conditions.

In the present study, to elucidate mechanisms of growth suppression in YIBO-pdc1/5Δ, we performed carbon metabolic flux analysis under micro-aerobic conditions. Our results indicate that growth suppression of YIBO-pdc1/5Δ is caused by decreased flux to the pentose phosphate pathway, which supplies ribose-5-phosphate, a precursor for histidine synthesis in Sacchar omyces cerevisiae. In addition, significant accumulation of pyruvate was observed in the continuous culture.

l-Anserine Increases Muscle Differentiation and Muscle Contractility in Human Skeletal Muscle Cells.

l-Anserine, an imidazole peptide, has a variety of physiological activities, but its effects on skeletal muscle differentiation and muscle contractile force remain unknown. Thus, in this study, we investigated the effect of l-anserine on muscle differentiation and muscle contractile force in human skeletal muscle cells. In two-dimensional culture, 1 µM l-anserine significantly increased the myotube diameters (26.5 ± 1.71, 27.7 ± 1.08, and 28.8 ± 0.85 µm with 0, 0.1, and 1 µM l-anserine, respectively) and the expression levels of genes involved in muscle differentiation and the sarcomere structure. In three-dimensional culture, 1 µM l-anserine significantly increased the contractile force of engineered human skeletal muscle tissues cultured on a microdevice (1.99 ± 0.30, 2.17 ± 0.62, 2.66 ± 0.39, and 3.28 ± 0.85 µN with 0, 0.1, 0.5, and 1 µM l-anserine, respectively). l-Anserine also increased the myotube diameters and the proportion of myotubes with sarcomere structures in the cultured tissues. Furthermore, the histamine receptor 1 (H1R) antagonist attenuated the l-anserine-induced increase in the contractile force, suggesting the involvement of H1R in the mechanism of action of l-anserine. This study showed for the first time that l-anserine enhances muscle differentiation and muscle contractility via H1R.

Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling.

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) differentiated into affected cell types can potentially recapitulate disease pathology more accurately than existing disease models. Such successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection, and clonal variations must be overcome. Moreover, their functionality should be carefully examined. Here, we demonstrated that bulk MYOD1-hiPSCs established with puromycin selection rather than G418 selection showed rapid and highly efficient differentiation. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties of clonally established MYOD1-hiPSCs, suggesting that it is possible to minimize clonal variations. Moreover, disease-specific hiPSCs of spinal bulbar muscular atrophy (SBMA) could be efficiently differentiated via this method into skeletal muscle that showed disease phenotypes, suggesting the applicability of this method for disease analysis. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical stimulation, indicating their functionality. Thus, our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and may facilitate the generation of muscular disease models.

Enhanced angiogenesis by transplantation of mesenchymal stem cell sheet created by a novel magnetic tissue engineering method.

OBJECTIVE: Therapeutic angiogenesis with cell transplantation represents a novel strategy for severe ischemic diseases. However, some patients have poor response to such conventional injection-based angiogenic cell therapy. Here, we investigated a therapeutic potential of mesenchymal stem cell (MSC) sheet created by a novel magnetite tissue engineering technology for reparative angiogenesis. METHODS AND RESULTS: Human MSCs incubated with magnetic nanoparticle-containing liposomes were cultured, and a magnet was placed on the reverse side. Magnetized MSCs formed multilayered cell sheets according to magnetic force. Nude mice were subjected to unilateral hind limb ischemia and separated into 3 groups. For the control group, saline was injected into ischemic tissue. In the MSC-injected group, mice received magnetized MSCs by conventional needle injections without sheet formula as a control cell group. In the MSC-sheet group, MSC sheet was layered onto the ischemic tissues before skin closure. Blood flow recovery and the extent of angiogenesis were assessed by a laser Doppler blood flowmetry and histological capillary density, respectively. The MSC-sheet group had a greater angiogenesis in ischemic tissues compared to the control and MSC-injected groups. The angiogenic and tissue-preserving effects of MSC sheets were attributable to an increased expression of vascular endothelial growth factor and reduced apoptosis in ischemic tissues. In cultured MSCs, magnetic labeling itself inhibited apoptosis via a catalase-like antioxidative mechanism. CONCLUSIONS: MSC sheet created by the novel magnetic nanoparticle-based tissue engineering technology would represent a new modality for therapeutic angiogenesis and tissue regeneration.