Large-scale cultivation of human iPS cells in bioreactor with reciprocal mixing.

A substantial number of human iPS cells (hiPSCs) is needed for cell therapy to be successful against various diseases. We previously reported on a bioreactor with reciprocal mixing that produces specific physical properties that differ from those of conventional bioreactors with rotary paddle stirring. Moreover, such reactors not only provide a homogeneous environment but also allow the control of spheroid size by changing the mixing speed. In this study, we applied this bioreactor to the large-scale cultivation of hiPSCs. Approximately 10 billion hiPSCs were obtained from 2.0 L of culture, and the high expression of pluripotency markers was maintained. Our findings indicate that a bioreactor with reciprocal mixing can be used for large-scale hiPSC cultivation.

Improved cultivation of Chinese hamster ovary cells in bioreactor with reciprocal mixing.

We have constructed a new bioreactor with reciprocal mixing that is better suited for the cultivation of delicate animal cells. In-silico simulation (computational fluid dynamics) suggested both maximum and average shear stresses in the bioreactor with reciprocal mixing to be remarkably lower than in a conventional bioreactor with rotary mixing. Although we could not find any difference in growth speed and cell density between the bioreactors with reciprocal and rotary mixing, we did find cell viability in the reciprocal-mixing bioreactor to be retained longer than in the rotary-paddle bioreactor. This implied that cell culture in a bioreactor with reciprocal mixing could be prolonged for the production of target proteins. Leakage of lactate dehydrogenase activity into the culture medium was suppressed much more in the reciprocal-mixing bioreactor than in the rotary-paddle one. Production of human tissue plasminogen activator in the former system was also observed to be much higher than in the latter. Therefore, a bioreactor with reciprocal mixing was concluded to be better suited for the cultivation of animal cells and efficient production of proteins, such as antibody drugs and various growth factors.

Cytoplasmic fusion between an enlarged embryonic stem cell and a somatic cell using a microtunnel device.

Based on a previous finding that fusion of a somatic cell with an embryonic stem (ES) cell reprogrammed the somatic cell, genes for reprogramming transcription factors were selected and induced pluripotent stem (iPS) cell technology was developed. The cell fusion itself produced a tetraploid cell. To avoid nuclear fusion, a method for cytoplasmic fusion using a microtunnel device was developed. However, the ES cell was too small for cell pairing at the device. Therefore, in the present study, ES cell enlargement was carried out with the colchicine derivative demecolcine (DC). DC induced enlargement of ES cells without loss of their stemness. When an enlarged ES cell was paired with a somatic cell in the microtunnel device, cytoplasmic fusion was observed. The present method may be useful for further development of reprogramming techniques for iPS cell preparation without gene transfection.

Micropatterned nanolayers immobilized with nerve growth factor for neurite formation of PC12 cells.

BACKGROUND: Nerve regeneration is important for the treatment of degenerative diseases and neurons injured by accidents. Nerve growth factor (NGF) has been previously conjugated to materials for promotion of neurogenesis. MATERIALS AND METHODS: Photoreactive gelatin was prepared by chemical coupling of gelatin with azidobenzoic acid (P-gel), and then NGF was immobilized on substrates in the presence or absence of micropatterned photomasks. UV irradiation induced crosslinking reactions of P-gel with itself, NGF, and the plate for immobilization. RESULTS: By adjustment of the P-gel concentration, the nanometer-order height of micropatterns was controlled. NGF was quantitatively immobilized with increasing amounts of P-gel. Immobilized NGF induced neurite outgrowth of PC12 cells, a cell line derived from a pheochromocytoma of the rat adrenal medulla, at the same level as soluble NGF. The immobilized NGF showed higher thermal stability than the soluble NGF and was repeatedly used without loss of biological activity. The 3D structure (height of the formed micropattern) regulated the behavior of neurite guidance. As a result, the orientation of neurites was regulated by the stripe pattern width. CONCLUSION: The micropattern-immobilized NGF nanolayer biochemically and topologically regulated neurite formation.

Antibacterial Properties of Silver Nanoparticles Embedded on Polyelectrolyte Hydrogels Based on α-Amino Acid Residues.

Polyelectrolyte hydrogels bearing l-phenylalanine (PHE), l-valine (AVA), and l-histidine (Hist) residues were used as scaffolds for the formation of silver nanoparticles by reduction of Ag⁺ ions with NaBH4. The interaction with the metal ion allowed a prompt collapse of the swollen hydrogel, due to the neutralization reaction of basic groups present on the polymer. The imidazole nitrogen of the hydrogel with Hist demonstrated greater complexing capacity with the Ag⁺ ion compared to the hydrogels with carboxyl groups. The subsequent reduction to metallic silver allowed for the restoration of the hydrogel's degree of swelling to the starting value. Transmission electron microscopy (TEM) and spectroscopic analyses showed, respectively, a uniform distribution of the 15 nm spherical silver nanoparticles embedded on the hydrogel and peak optical properties around a wavelength of 400 nm due to the surface plasmonic effect. Unlike native hydrogels, the composite hydrogels containing silver nanoparticles showed good antibacterial activity as gram+/gram- bactericides, and higher antifungal activity against S. cerevisiae.

Characterization of Giant Modular PKSs Provides Insight into Genetic Mechanism for Structural Diversification of Aminopolyol Polyketides.

Polyketides form many clinically valuable compounds. However, manipulation of their biosynthesis remains highly challenging. An understanding of gene cluster evolution provides a rationale for reprogramming of the biosynthetic machinery. Herein, we report characterization of giant modular polyketide synthases (PKSs) responsible for the production of aminopolyol polyketides. Heterologous expression of over 150 kbp polyketide gene clusters successfully afforded their products, whose stereochemistry was established by taking advantage of bioinformatic analysis. Furthermore, phylogenetic analysis of highly homologous but functionally diverse domains from the giant PKSs demonstrated the evolutionary mechanism for structural diversification of polyketides. The gene clusters characterized herein, together with their evolutionary insights, are promising genetic building blocks for de novo production of unnatural polyketides.

Serum-free culturing of human mesenchymal stem cells with immobilized growth factors.

In vitro expansion of human mesenchymal stem cells (hMSCs) using serum-free culture medium is important for basic research and clinical applications. It is known that some growth factors are required for developing a defined medium for hMSC culture. However, growth factors usually show poor stability, short circulating half-life and a rapid rate of cellular internalization when they are in a diffusible state. A potential way to overcome these problems is to immobilize growth factors on materials. Here, three different types of growth factors, basic fibroblast growth factor, transforming growth factor-beta, and platelet-derived growth factor, were co-immobilized on cell culture dish surfaces by photo-reactive gelatin and used for serum-free hMSC cultures. The results showed that the immobilized growth factors supported cell proliferation similarly to the serum-containing medium. More importantly, the immobilization of growth factors significantly improved their thermal stability and efficiently prolonged their shelf life at 4 °C and 37 °C. Furthermore, the immobilized growth factors could be reused at least three times without losing their stimulation effect on cell proliferation. This photo-reactive gelatin-based immobilization of growth factors appears to be a promising method for serum-free hMSC culturing.

Enhancement of synergistic gene silencing by RNA interference using branched "3-in-1" trimer siRNA.

Nanostructured RNA carrying three different siRNAs was assembled to silence three target genes (Axin, APC, and GSK-3ß) in the Wnt/ß-catenin signaling pathway. The trimer RNA nanostructure included equimolar concentrations of three oligonucleotide sequences. The three armed structures and the size of the trimer RNA were confirmed by agarose gel electrophoresis, atomic force microscopy, and dynamic light scattering. In the presence of 10% human serum, the trimer RNA was able to resist degradation and maintained an intact structure for more than two hours. Protein expression analyses showed specific repression of the target proteins by siRNAs. As a result, the expression of luciferase in a ß-catenin reporter vector was significantly increased by the trimer RNA compared with a pool of the three individual siRNAs. This high activity at a low concentration was considered to be due to the 3-in-1 format of the trimer and the long-term resistance to serum proteins by nanostructure formation. We demonstrated that a nanostructured "3-in-1" siRNA is effective in enhancing the effect of RNA interference.

Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast.

Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4(+) and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

Structures and biological activities of azaphilones produced by Penicillium sp. KCB11A109 from a ginseng field.

Twelve metabolites, including five highly oxygenated azaphilones, geumsanols A-E, along with seven known analogues were isolated from Penicillium sp. KCB11A109, a fungus derived from a ginseng field. Their structures were assigned by spectroscopic means (NMR and MS), and stereochemistries were determined by extensive spectroscopic analyses ((1)H-(1)H coupling constants, NOESY, and HETLOC) and chemical derivatizations (modified Mosher's method and acetonide formation). The isolates were evaluated for their anticancer, antimicrobial, antimalarial activities, and phenotypic effects in zebrafish development. Of these compounds possessing no pyranoquinone core, only geumsanol E exhibited cytotoxic activities and toxic effects on zebrafish embryos, suggesting that a double bond at C-11 and C-12 is important for biological activity.

Boseongazepines A-C, pyrrolobenzodiazepine derivatives from a Streptomyces sp. 11A057.

Three new pyrrolobenzodiazepine derivatives, boseongazepines A-C (1-3), were isolated from a culture broth of Streptomyces sp. 11A057, together with the known compound usabamycin B (4). The structures of 1-4 were determined through the analysis of spectroscopic data including extensive 1D-, 2D-NMR, and MS techniques. Cell growth inhibition effects of these compounds were evaluated against Jurkat, K-562, HL-60, and HepG2 cell lines.

Isolation of new polyketide metabolites, linearolides A and B, from Streptomyces sp. RK95-74.

Although all Streptomyces strains are now thought to have 20-30 gene clusters for secondary metabolite biosynthesis, we cannot actually identify so many kinds of metabolites from one strain by conventional methods. Using Streptomyces sp. RK95-74, previously found as a cytotrienin producer, we searched new metabolites other than cytotrienin derivatives. Following the cultivation with new media and the peak-guided fractionation, we have found new compounds with new polyketide scaffold, named linearolides A and B.

Terpendole E, a kinesin Eg5 inhibitor, is a key biosynthetic intermediate of indole-diterpenes in the producing fungus Chaunopycnis alba.

Terpendole E is the first natural product inhibitor of kinesin Eg5. Because terpendole E production is unstable, we isolated and analyzed the terpendole E biosynthetic gene cluster, which consists of seven genes encoding three P450 monooxygenases (TerP, TerQ, and TerK), an FAD-dependent monooxygenase (TerM), a terpene cyclase (TerB), and two prenyltransferases (TerC and TerF). Gene knockout and feeding experiments revealed that terpendole E is a key intermediate in terpendole biosynthesis and is produced by the action of the key enzyme TerQ from paspaline, a common biosynthetic intermediate of indole-diterpenes. TerP converts terpendole E to a downstream intermediate specific to terpendole biosynthesis and converts paspaline to shunt metabolites. We successfully overproduced terpendole E by disrupting the terP gene. We propose that terpendole E is a key biosynthetic intermediate of terpendoles and related indole-diterpenes.

Biochemical characterization of a novel indole prenyltransferase from Streptomyces sp. SN-593.

Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (-)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 min(-1) microM(-1)) for L-Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl-L-Trp synthase (6-DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, DeltaiptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl-L-Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde.

Deamino-hydroxy-phoslactomycin B, a biosynthetic precursor of phoslactomycin, induces myeloid differentiation in HL-60 cells.

During the screening for novel differentiation inducers, we found that a culture broth of Streptomyces sp. HK-803 induced myeloid differentiation of HL-60 cells. The active substance was identified as deamino-hydroxy-phoslactomycin B (HPLM) by mass spectrometry, and synthesized HPLM also induced the differentiation of HL-60 cells. HPLM showed greater inhibition of protein phosphatase 2A (PP2A) activity than phoslactomycin B (PLMB); however, PLMB and okadaic acid did not induce differentiation. Moreover, treatment with ATRA and 1alpha, 25(OH)2D3 induced retinoic acid receptor-beta and 1alpha, 25(OH)2D3 24-hydroxylase, respectively, whereas HPLM did not, suggesting that HPLM is a novel differentiation inducer.

The effect of mechanical loading on the metabolism of growth plate chondrocytes.

It is well known that mechanical loading influences the endochondral bone formation essential for the growth and development of longitudinal bones. The question was, however, asked whether the effect of mechanical loading on the chondrocyte metabolism is dependent on the loading frequency. This study was aimed at evaluating the effect of tensile loadings with various frequencies on the proliferation of growth plate chondrocytes and extracellular matrix synthesis. The chondrocytes obtained from rib growth plate cartilage of 4-week-old male Wistar strain rats were cultured by day 4 and day 11 and used as proliferating and matrix-forming chondrocytes, respectively. Intermittent tensile stresses with different frequencies were applied to each stage chondrocyte. DNA syntheses were examined by measuring the incorporation of [(3)H]thymidine into the cells. Furthermore, the rates of collagen and proteoglycan syntheses were determined by measuring the incorporation of [2,3-(3)H]proline and [(35)S]sulfate into the cells, respectively. At the proliferating stage, intermittent tensions with the frequencies of 30 cycles/min and 150 cycles/min significantly (p < 0.05) upregulated the syntheses of DNA, which indicates the promotion of chondrocyte proliferation. At the matrix-forming stage, collagen, and proteoglycan syntheses also enhanced with increase of the loading frequency. In particular, the intermittent tension with the frequencies of 30 cycles/min and 150 cycles/min increased significantly (p < 0.05 or p < 0.01) both the collagen and proteoglycan syntheses. These results suggest that the proliferation and differentiation of growth plate chondrocytes are regulated by the mechanical loading and that the chondrocyte metabolism enhanced with increase of loading frequency. These may give more insight into the possible mechanism leading to endochondral bone formation.

Inhibition of the proliferation of human periodontal ligament fibroblasts by hyaluronidase.

Hyaluronan (HA) exists in various living tissues as one of the major matrix macromolecules, and is well known to play an integral role in cell differentiation and proliferation. The present study was conducted to elucidate whether or not the proliferation of periodontal ligament (PDL) cells are affected specifically by the degradation of HA by hyaluronidasze (HAase). Human PDL fibroblasts were isolated and cultured with and without 15-150U/ml bovine testicular HAase from 1 to 11 days after seeding. The cells were also cultured with anti-CD44 antibody of 2 microg/ml. For the control against the anti-CD44 antibody treatment, 2 microg/ml IgG was used. The HA-dependent pericellular matrix was visualized by particle-exclusion assay. The number of cells was counted by MTT assay during the proliferation. The mRNA levels of HA synthases (HASs), HAases (HYALs) and CD44s were examined by a quantitative real-time PCR analysis. The cell proliferation was inhibited by the treatment with HAase and anti-CD44 antibody in cultured PDL fibroblasts. HASs mRNAs were down-regulated, whereas HYALs mRNAs were up-regulated significantly by the treatment with HAase and anti-CD44 antibody. The CD44s mRNA level exhibited no significant changes. These results suggest that HA may contribute to modulate the proliferation of cultured human PDL cells through a CD44-mediated mechanism.

Characterization of short-chain poly3-hydroxybutyrate in baker's yeast.

A short-chain poly3-hydroxybutyrate including four comonomers, originating from a complex with calcium polyphosphate, was isolated from commercial baker's yeast cells (Saccharomyces cerevisiae) and characterized as the second complexed poly(3-hydroxyalkanoate) (cPHA) in eukaryotes. The number-average molecular weight of 4982.5 Da with a polydispersity index of 1.11 was much lower than that of beet cPHA previously isolated. End-group analysis suggested that at least 60% of the molecules form the cyclic structures. Here, the organism-dependent structural diversity of cPHAs was completely established. It was also found that a change of culture medium influences the molecular weight but not the polydispersity of baker's yeast cPHA.

Enzymatic generation of the antimetabolite gamma,gamma-dichloroaminobutyrate by NRPS and mononuclear iron halogenase action in a streptomycete.

Four adjacent open reading frames, cytC1-C4, were cloned from a cytotrienin-producing strain of a Streptomyces sp. by using primers derived from the conserved region of a gene encoding a nonheme iron halogenase, CmaB, in coronamic acid biosynthesis. CytC1-3 were active after expression in Escherichia coli, and CytC4 was active after expression in Pseudomonas putida. CytC1, a relatively promiscuous adenylation enzyme, installs the aminoacyl moieties on the phosphopantetheinyl arm of the holo carrier protein CytC2. CytC3 is a nonheme iron halogenase that will generate both gamma-chloro- and gamma,gamma-dichloroaminobutyryl-S-CytC2 from aminobutyryl-S-CytC2. CytC4, a thioesterase, hydrolytically releases the dichloroaminobutyrate, a known streptomycete antibiotic. Thus, this short four-protein pathway is likely the biosynthetic source of this amino acid antimetabolite. This four-enzyme system analogously converts the proS-methyl group of valine to the dichloromethyl product regio- and stereospecifically.