Three-dimensional co-culture model employing silica nonwoven fabrics to enhance cell-to-cell communication of paracrine signaling between hepatocytes and fibroblasts.

The realization that soluble factors secreted by heterotypic cells play an importanta role in paracrine signaling, which facilitates intercellular communication, enabled the development of physiologically relevant co-culture models for drug screening and the engineering of tissues, such as hepatic tissues. The most crucial issues confronting the use of conventional membrane inserts in segregated co-culture models that are used to study paracrine signaling between heterotypic cells have been identified as long-term viability and retention of cell-specific functions, especially when isolated primary cells are used. Herein, we present an in vitro segregated co-culture model consisting of a well plate incubated with rat primary hepatocytes and normal human dermal fibroblasts which were segregated using a membrane insert with silica nonwoven fabric (SNF) on it. SNF, which mimics a physiological environment much more effectively than a two-dimensional (2D) one, promotes cell differentiation and resultant paracrine signaling in a manner that is not possible in a conventional 2D culture, owing to high mechanical strength generated by its inorganic materials and interconnected network structure. In segregated co-cultures, SNF clearly enhanced the functions of hepatocytes and fibroblasts, thereby showing its potential as a measure of paracrine signaling. These results may advance the understanding of the role played by paracrine signaling in cell-to-cell communication and provide novel insights into the applications of drug metabolism, tissue repair, and regeneration.

Heterogeneous Ganglioside-Enriched Nanoclusters with Different Densities in Membrane Rafts Detected by a Peptidyl Molecular Probe.

The specific features of the lateral distribution of gangliosides play key roles in cell-cell communications and the onset of various diseases related to the plasma membrane. We herein demonstrated that an artificial peptide identified from a phage-displayed library is available as a molecular probe for specific ganglioside nanoclustering sites in caveolae/membrane rafts on the cell surface. Atomic force microscopy studies indicated that the peptide specifically binds to the highly enriched monosialoganglioside GM1 nanodomains of reconstituted lipid bilayers composed of GM1, sphingomyelin, cholesterol, and unsaturated phospholipids. The ganglioside-containing area recognized by the peptide on the surface of PC12 cells was part of the area recognized by the cholera toxin B subunit, which has high affinity for GM1. Furthermore, the peptide bound to the cell surface after a treatment with methyl-ß-cyclodextrin (MßCD), which disrupts membrane rafts by removing cholesterol. The present results indicate that there are heterogeneous ganglioside clusters with different ganglioside densities in caveolae/membrane rafts, and the peptidyl probe selectively recognizes the high-density ganglioside nanodomain that resists the MßCD treatment. This peptidyl probe will be useful for obtaining information on the lipid organization of the cell membrane and will help clarify the mechanisms by which the lateral distribution of gangliosides affects biological functions and the onset of diseases.

Improvement of Hepatic Functions by Spheroids Coculture with Fibroblasts in 3D Silica Nonwoven Fabrics.

In order to realize organ-on-a-chip as an effective tool for regenerative medicine and drug development, tissue-mimic cell culture methods which promote liver-specific function for long period have been developed. We have previously demonstrated that coculture of hepatocyte spheroids on fibroblasts using micropatterned substrate improved the hepatic functions due to the heterotypic cell-cell interactions and paracrine signaling from each other. In addition, hepatocyte function cultured as monolayer was also promoted in separately coculture with fibroblasts cultured as monolayer, and it is more improved in separately coculture with fibroblasts in 3D silica nonwoven fabrics. In this study, separately coculture of hepatocyte spheroids with fibroblasts cultured on 3D silica nonwoven fabrics was estimated for further improvement of hepatocyte functions. The hepatic function cocultured with fibroblast was more promoted than mono spheroids culture. The functional enhancement was significantly most improved in separately coculture with fibroblast in 3D silica nonwoven fabrics. Thus, these results were suggested that 3D culture of fibroblasts in 3D silica nonwoven fabrics increased the heterotypic secretion of paracrine factors, and it is essential for improved hepatic performance.

Noninvasive Fingerprinting-Based Tracking of Replicative Cellular Senescence Using a Colorimetric Polyion Complex Array.

A fingerprint-based sensing approach was used to characterize in vitro cellular senescence. Secretion profiles of cultured human fibroblasts in different senescent stages were transformed into colorimetric enzyme-activity fingerprints by applying cell culture media to a polyion complex array. Analysis of the obtained fingerprints using pattern recognition methods, such as linear discriminant analysis and hierarchical clustering analysis, revealed that the polyion complex array allows the noninvasive tracking of the replicative senescence progress even in those stages where a conventional marker such as senescence-associated ß-galactosidase is negative. This fingerprint-based approach should thus offer an effective way for the routine monitoring or screening of in vitro cell senescence studies.

Effect of protein adsorption layers and solution treatments on hydroxyapatite deposition on polystyrene plate surfaces in simulated body fluids.

We have developed a method to functionalize polystyrene (PS) cell culture plates with hydroxyapatite (HAp) via protein adsorption layers such as human serum albumin (HSA) in simulated body fluids (SBFs). In order to investigate the versatility the method, in this study the effect of protein adsorption layers on HAp deposition on PS plate surfaces in SBF was evaluated. Pretreatments with alternate soaking process (ASP) using solutions containing calcium ions and phosphate ions followed by incubation with SBF for 24 h resulted in HAp deposition on PS plates with adsorption layers of HSA, type I collagen, hen egg white lysozyme, and poly L-glutamic acid, an acidic protein analogue: the deposition behaviors were correlated with adsorption ability and charge state of proteins. We also demonstrated that commercially available tissue culture-treated PS (TCPS) were directly coated with bone-like HAp using the same ASP and SBF processes. Human mesenchymal stem cells adhered and stretched on the HAp-coated TCPS plates in a similar manner to the case of the HAp-coated PS plates prepared via HSA adsorption layers. The results indicate that the present methods are useful for preparing bone-like HAp-coated cell culture plates that can be utilized function of adsorbed proteins and that are obtainable conveniently and at low-cost.

Hot-Press-Assisted Adhesions between Polyimide Films and Titanium Plates Utilizing Coating Layers of Silane Coupling Agents.

The development of low material-consuming adhesion techniques for different kinds of materials such as polymers and metals is important for the realization of sustainable societies. This study demonstrates that coating layers, expected to be formed as self-assembled monolayers, of silane coupling agents can act as adhesion layers at the polymer film-metal plate interfaces. Polyimide films were alkaline hydrolyzed to generate carboxy groups on their surfaces, whereas titanium plate surfaces were treated with the aminosilanes to form their coating layers thereon. These modified surfaces were placed in contact with each other and then hot pressed, which resulted in adhesion between them. An examination of the adhesion strength using lap shear tests and surface characterization of the prepared surfaces using X-ray photoelectron spectroscopy and other techniques indicated the formation of ionic bonds and/or amide bonds between the carboxy groups of the PI film surfaces and the amino groups immobilized on the titanium plate surfaces. The activation of the carboxy groups using N-hydroxysuccinimide resulted in adhesion obtaining a water-resistant property, which supported the increase in amide bond formation. On the basis of the results, the adhesion mechanism and the possible breaking points upon the breaking of adhesions are proposed.

[Analysis of gene expression and DNA methylation patterns in childhood acute lymphoblastic leukemia].

The 5-year survival rate of patients with childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) now exceeds 90%, though there are still patients who fail to achieve remission or relapse early. To improve the outcomes of these cases, new diagnostic markers for stratification of those with unfavorable outcomes and novel targets for treatment have been investigated based on data from the OMICs analysis. We performed gene expression analysis of leukemic cells from 91 near-diploid BCP-ALL cases without specific fusion genes enrolled in Tokyo Children's Cancer Study Group (TCCSG)-L0416 & L0616 clinical trials employing the Affymetrix Human Genome U133 Plus 2.0 Array. Among them, DNA methylation status was analyzed in 24 cases by using the Infinium HumanMethylation450 BeadChip. Herein, initially, the current situations of gene expression analysis and DNA methylation analysis of childhood BCP-ALL are reviewed. Then, our analyses of gene expressions and DNA methylation related to the prognosis of childhood ALL without fusion genes are presented.

ATF7IP as a novel PDGFRB fusion partner in acute lymphoblastic leukaemia in children.

We identified ATF7IP as a novel PDGFRB fusion partner in B-progenitor acute lymphoblastic leukaemia (B-ALL) and showed that B-ALL with ATF7IP/PDGFRB translocation is included within the genomic lesions of a Philadelphia chromosome (Ph)-like ALL subgroup. Comprehensive analyses of previous repositories of gene expression data sets disclosed that B-ALL cases with high PDGFRB expression level in the context of the Ph-like ALL gene are likely to have a PDGFRB translocation. Thus, it is possible that measurement of the PDGFRB expression level can be utilized as a screening test for the detection of the cryptic PDGFRB translocation, especially within the Ph-like ALL subgroup.

Poor responses to tyrosine kinase inhibitors in a child with precursor B-cell acute lymphoblastic leukemia with SNX2-ABL1 chimeric transcript.

In addition to BCR, various rare fusion partners for the ABL1 gene have been reported in leukemia. We have identified the fusion gene SNX2-ABL1 in a pediatric case of acute lymphoblastic leukemia (ALL), which has only once previously been reported in an adult patient. Cytogenetic analysis detected this fusion gene arising from a t(5;9)(q22;q34) translocation. ALL cells carrying a SNX2-ABL1 fusion exhibited a BCR-ABL1+ ALL-like gene expression profile. The patient poorly responded to dasatinib but partially responded to imatinib. Treatment using tyrosine kinase inhibitors requires further investigation to optimize the genotype-based treatment stratification for patients with SNX2-ABL1 fusion.

Microfabrication of Gelatin Methacrylate/Hydroxyapatite Composites by Utilizing Alternate Soaking Process.

To construct a complex three-dimensional (3D) structure mimicking bone microstructure, hydrogel models of polymerized gelatin methacrylate (pGelMA) were fabricated by using stereolithography and modified with hydroxyapatite (HAp) via an alternate soaking process (ASP) using a solution of calcium and phosphate ions. Fabricated pGelMA line models whose widths were designed as 100, 300, and 600 µm were modified with HAp by ASP by changing the immersion time and number of cycles. After ASP, all of the line models with widths of 100, 300, and 600 µm were successfully modified with HAp, and large amounts of HAp were covered with the fabricated models by increasing both the immersion time and the number of cycles in ASP. HAp was observed near the surface of the line model with a width of 600 µm after ASP at an immersion time of 10 s, while the entire model was modified with HAp using ASPs for longer immersion times. The adhesion and spread of mesenchymal stem cells (MSCs) on the pGelMA-HAp discs depended on the ASP conditions. Moreover, the HAp modification of 3D pyramid models without alteration of the microstructure was also conducted. This two-step fabrication method of first fabricating frameworks of hydrogel models by stereolithography and subsequently modifying the fabricated models with HAp will lead to the development of 3D cell culture systems to support bone grafts or to create biological niches, such as artificial bone marrow.

Evaluation of the immunotoxicity potential of nanomaterials using THP-1 cells.

With the expansion of nanomaterials (NMs) usage, concerns about their toxicity are increasing, and the wide variety of NMs makes it difficult to assess their toxicity. Therefore, the development of a high-throughput, accurate, and certified method to evaluate the immunotoxicity of NMs is required. In this study, we assessed the immunotoxicity potential of various NMs, such as nanoparticles of silver, silica, and titanium dioxide, using the human Cell Line Activation Test (h-CLAT) at the cellular level. After exposure to silver nanoparticle dispersions, the expression levels of CD86 and CD54 increased, suggesting the activation of antigen-presenting cells (APCs) by silver nanoparticles. Quantification of silver ions eluted from silver nanoparticles and the activation of APCs by silver ions suggested that it was due to the release of silver ions. Silica nanoparticles also increased the expression of CD86 and/or CD54, and their activation ability correlated with the synthesis methods and hydrodynamic diameters. The ability of titanium dioxide to activate APCs differed depending on the crystal type and hydrodynamic diameter. These results suggest a potential method to evaluate the immunotoxicity potential of various NMs based on their ability to activate APCs using human monocytic THP-1 cells. This method will be valuable in assessing the immunotoxicity potential and elucidating the immunotoxic mechanisms of NMs.

The human CD10 lacking an N-glycan at Asn(628) is deficient in surface expression and neutral endopeptidase activity.

BACKGROUND: CD10, also known as neprilysin or enkephalinase exhibiting neutral endopeptidase (NEP) activity, is expressed by B-lineage hematopoietic cells as well as a variety of cells from normal tissues. It cleaves peptides such as cytokines to act for terminating inflammatory responses. Although CD10 molecules of the human pre-B-cell line NALM-6 have 6 consensus N-glycosylation sites, three of them are known to be N-glycosylated by X-ray crystallography. METHODS: In order to investigate the role of N-glycans in the full expression of NEP activity, we modified N-glycans by treatment of NALM6 cells with various glycosidases or alter each of the consensus N-glycosylation sites by generating site-directed mutagenesis and compared the NEP activities of the sugar-altered CD10 with those of intact CD10. RESULTS: CD10 of the human B-cell line NALM-6 was dominantly localized in raft microdomains and heterogeneously N-glycosylated. Although neither desialylation nor further degalactosylation caused defective NEP activity, removal of only a small part of N-glycans by treatment with glycopeptidase F under non-denaturing conditions decreased NEP activity completely. All of the three consensus sites of CD10 in HEK293 cells introduced with wild type-CD10 were confirmed to be N-glycosylated. Surface expression of N-glycan at Asn(628)-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities. CONCLUSIONS: N-glycosylation at Asn(628) is essential not only for NEP activities, but also for surface expression. GENERAL SIGNIFICANCE: Quality control system does not allow dysfunctional ecto-type proteases to express on plasma membrane.

ZNF385B is characteristically expressed in germinal center B cells and involved in B-cell apoptosis.

We previously identified zinc finger (ZF) protein ZNF385B as a molecule specifically expressed in Burkitt's lymphoma (BL) among hematologic malignancies. Here, we investigated ZNF385B expression in healthy B cells in a variety of hematological tissues by RT-PCR and immunohistochemistry. ZNF385B expression was found to be limited to a subset of GC B cells, the healthy counterpart to BL B cells. To elucidate the function of ZNF385B in healthy B cells, we established a tetracycline-controlled protein-inducible system in B-cell lines and observed that ectopic expression of the longest transcript variant of ZNF385B, possessing four ZF domains, induced upregulation of PERP and FAS/CD95, a downstream target of p53, and activation of caspase, resulting in apoptosis induction. However, a ZNF385B deletion mutant with three ZF domains corresponding to shorter isoforms, did not induce upregulation; rather it inhibited apoptosis induced by CD20 cross-linking and BCR stimulation. The direct binding of ZNF385B with p53 has suggested the involvement of ZNF385B in B-cell apoptosis via modulation of p53 transactivation; our data indicate that ZNF385B characteristically expressed in GC B cells has both proapoptotic and antiapoptotic activities depending on the type of isoform and should be a novel player in GC B-cell selection.

Density of GM1 in nanoclusters is a critical factor in the formation of a spherical assembly of amyloid ß-protein on synaptic plasma membranes.

The deposition of amyloid ß-protein (Aß) is a pathological hallmark of Alzheimer's disease (AD). We previously found that the ganglioside-enriched microdomains (ganglioside clusters) in presynaptic neuronal membranes play a key role in the initiation of the Aß assembly process. However, not all ganglioside clusters accelerate Aß assembly. In the present study, we directly observed a spherical Aß in an atomic force microscopic study on the morphology of a reconstituted lipid bilayer composed of lipids that were extracted from a detergent-resistant membrane microdomain (DRM) fraction of synaptosomes prepared from aged mouse brain. The Aß assembly was generated on a distinctive GM1 domain, which was characterized as the Aß-sensitive ganglioside nanocluster (ASIGN). By using an artificial GM1 cluster-binding peptide, ASIGN was found to have a high density of GM1; therefore, there would be a critical density of GM1 in nanoclusters to induce Aß binding and assembly. These results suggest that ganglioside-bound Aß (GAß), which acts as an endogenous seed for Aß fibril formation in AD brains, is generated on ASIGN on synaptosomal membranes.

Incorporation of glycosylated amino acid into protein by an in vitro translation system.

O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.

Specific ion effects on the aggregation of polysaccharide-based polyelectrolyte complex particles induced by monovalent ions within Hofmeister series.

Polysaccharide-based polyelectrolyte complex (PEC) particles have been utilized as carriers for drug delivery systems (DDS) and as building components for material development. Despite their versatility, the aggregation mechanism of PEC particles in the presence of salts remains unclear. To clarify the aggregation mechanism, the specific ion effects of monovalent salts within the Hofmeister series on the aggregation behavior of PEC particles composed of chitosan and chondroitin sulfate C, which are often used as DDS carriers and materials, were studied. Here, we found that weakly hydrated chaotropic anions promoted the aggregation of positively charged PEC particles. The hydrophobicity of the PEC particles was increased by these ions. Strongly hydrated ions such as Cl- are less likely to accumulate in these particles, whereas weakly hydrated chaotropic ions such as SCN- are more likely to accumulate. Molecular dynamics simulations suggested that the hydrophobicity of PECs might be strengthened by ions due to changes in intrinsic and extrinsic ion pairs and hydrophobic interactions. Based on our results, it is expected that the control of surface hydrophilicity or hydrophobicity is an effective approach for controlling the stability of PEC particles in the presence of ions.

Preparation of Mechanically Anisotropic Polysaccharide Composite Films Using Roll-Press Techniques.

Natural polysaccharides are biocompatible and biodegradable; therefore, they can be used as feedstock for biodegradable structural materials and biomaterials. In this study, anisotropic polysaccharide composite films consisting of chondroitin sulfate C (CS) and chitosan (CHI) were fabricated from their polyion complex (PIC) gels by roll-press techniques. The obtained films (CS/CHI films) were thin and transparent, similar to the composite films prepared by hot-press techniques. The roll-press conditions were optimized, and it was observed that the molecular weight of CHI did not significantly affect the formability of the films, whereas the roll temperature and rolling speed were important. The tensile tests of the roll-pressed films revealed that the mechanical strength of the films in the mechanical direction (MD) was approximately 5 times higher than that in the transverse direction (TD), indicating that the roll-press techniques imparted mechanical anisotropy to the films. Additionally, the films shrank in the MD and expanded in the TD after immersion in aqueous solutions, followed by drying. Such anisotropic shrinking and expanding properties indicate that these films can be used as shape-memory materials.

Analysis of the aggregation mechanism of chondroitin sulfate/chitosan particles and fabrication of hydrogel cell scaffolds.

In this study, the aggregation mechanism of polyion complex (PIC) particles from chitosan (CHI) and chondroitin sulfate C (CS) in phosphate-buffered saline (PBS) was analyzed, and a novel method for the fabrication of hydrogels via aggregation was developed. The PBS induced a decrease in the ζ-potential of the CS/CHI PIC particles, increase in their diameter, and aggregation in a concentration-dependent manner. The hydrogels prepared by mixing CS/CHI PIC particle dispersion and PBS showed the PIC components, with porous structure, high swelling ratio (161.4 ± 13.3%), and high storage moduli (26.2 ± 1.4 kPa). By mixing PBS with suspended adhesive cells and CS/CHI PIC particle dispersion, hydrogels with high cell-loading efficiency were successfully synthesized. The loaded cells within the hydrogels exhibited high viability, uniform distribution, and formation of cell aggregates. These results indicate that CS/CHI-based hydrogels have a potential application as three-dimensional scaffolds for cell culture in tissue engineering.

Surface modification of silica nonwoven fabrics for osteogenesis of bone marrow-derived mesenchymal stem cells.

Silica nonwoven fabrics (SNFs) with high mechanical strength and porosity are known to exhibit high cell proliferation and osteogenic differentiation potential of mesenchymal stem cells (MSCs) by morphologically mimicking the extracellular matrix (ECM). To further improve the osteoinductive ability of SNFs, it could be effective to increase the interaction between MSCs and ECM components because exogenous ECM components seem to modulate the fate of MSCs differentiation. In this study, we developed immobilization methods for ECM components, such as collagen, fibronectin, and chondroitin sulphate C on SNFs, to improve cell-matrix interactions and examined their suitability for bone tissue regeneration. Collagen and fibronectin were immobilized via physical adsorption and chondroitin sulphate C was also immobilized by the layer-by-layer method combined with chitosan on SNF surfaces to maintain the high porosity of SNFs. The treated SNFs were characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. In osteogenic differentiation culture, modified SNFs showed significantly increased expression of osteogenic differentiation marker genes compared to unmodified SNFs. These results suggest that the present methods improve cell-matrix interactions and enhance the cellular functions of MSCs. We are convinced that these simple modification techniques for ECM components are effective in functionalizing various 3D fabric scaffolds possessing hydrophilic groups.

Hydroxyapatite Particles from Simulated Body Fluids with Different pH and Their Effects on Mesenchymal Stem Cells.

Bone-like hydroxyapatite (HAp) has been prepared by biomimetic synthesis using simulated body fluid (SBF), mimicking inorganic ion concentrations in human plasma, or 1.5SBF that has 1.5-times higher ion concentrations than SBF. In this study, the controllable preparations of HAp particles from 1.5SBF with different pH values were examined. The particles obtained as precipitates from 1.5SBF showed different morphologies and crystallinities depending on the pH of 1.5SBF. Micro-sized particles at pH 7.4 of 1.5SBF had a higher Ca/P ratio and crystallinity as compared with nano-sized particles at pH 8.0 and pH 8.4 of 1.5SBF. However, a mixture of micro-sized and nano-sized particles was obtained from pH 7.7 of 1.5SBF. When Ca2+ concentrations in 1.5SBF during mineralization were monitored, the concentration at pH 7.4 drastically decreased from 12 to 24 h. At higher pH, such as 8.0 and 8.4, the Ca2+ concentrations decreased during pH adjustment and slightly decreased even after 48 h. In this investigation at pH 7.7, the Ca2+ concentrations were higher than pH 8.0 and 8.4.Additionally, cytotoxicity of the obtained precipitates to mesenchymal stem cells was lower than that of synthetic HAp. Controllable preparation HAp particles from SBF has potential applications in the construction of building components of cell scaffolds.