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Hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells are potent cells to study individual-specific hepatotoxicity for drug screening test. However, the functions of metabolic enzymes are practically low. Here, we reconstituted stable and compact 3D spheroids of commercially available cryopreserved HLCs by our original spheroid formation method with high viscous methylcellulose medium. 3D formation enhanced the hepatic functions and maintained the functions for 14 days. Especially, the expression of cytochrome P450s was 10- to 100-fold enhanced compared to conventional 2D culture, which is applicable to a typical drug-metabolizing test using liquid chromatograph-tandem mass spectrometer. In conclusion, we successfully formed human HLC spheroid from commercially available cryo-preserved cells, which realized remarkable hepatic maturation by prolonged 3D culture, especially in terms of drug-metabolizing enzymes. Our spheroid formation technology has the potential to make HLC spheroids a potent tool in aspects of pharmaceutical research, such as drug screening and pharmacokinetic studies.

Hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells are potent cells to study individual-specific hepatotoxicity for drug screening test. However, the functions of metabolic enzymes are practically low. 3D spheroid is a potent strategy to enhance the differentiation efficacy from undifferentiated cells, but 3D formation of “differentiated” HLCs is not fully examined. Here, we successfully reconstituted stable and compact 3D spheroids of commercially available cryo-preserved HLCs by our original spheroid formation method with high viscous methylcellulose medium, while conventional formation methods never formed the spheroid. 3D formation enhanced the hepatic functions and maintained the functions for 14 days. Especially, drug-metabolizing enzymes, cytochrome P450, were 10- to 100-fold enhanced compared to conventional 2D culture, which is applicable to a typical drug-metabolizing test using LC-MS/MS. Our formation method has an impact on functional spheroid formation.

Dendritic spines have stable filamentous actin (F-actin) and dynamic F-actin. The formation of stable F-actin plays a pivotal role in spine formation. Drebrin binds to and stabilizes F-actin in dendritic spines. Interestingly, the conversion of the drebrin E isoform to drebrin A occurs in parallel with synapse formation, suggesting that this conversion promotes synapse formation via F-actin accumulation. In this study, we measured the dynamics of GFP-tagged drebrin E (GFP-DE) and drebrin A (GFP-DA) in cultured hippocampal neurons by fluorescence recovery after photobleaching analysis. We found that GFP-DA has a larger stable fraction than GFP-DE. The stable drebrin fraction reflects its accumulation in dendritic spines, therefore the isoform conversion may increase the amount of stable F-actin in dendritic spines. The stable fraction was dependent on the drebrin A-specific sequence "Ins2", located in the middle of the drebrin protein. In addition, F-actin depolymerization with latrunculin A significantly reduced the stable GFP-DA fraction. These findings indicate that preferential binding of drebrin A to F-actin than drebrin E causes higher stable fraction of drebrin A in dendritic spines, although the F-actin-binding ability of purified drebrin E and drebrin A are comparable. Therefore, we suggest that a drebrin isoform conversion from drebrin E to drebrin A in dendritic spines results in the accumulation of drebrin-bound stable F-actin, which plays a pivotal role in synapse formation.

Culture systems for 3-dimensional tissues, such as multicellular spheroids, are indispensable for high-throughput screening of primary or patient-derived xenograft (PDX)-expanded cancer tissues. Oxygen supply to the center of such spheroids is particularly critical for maintaining cellular functions as well as avoiding the development of a necrotic core. In this study, we evaluated 2 methods to enhance oxygen supply: (1) using culture plate with gas-permeable polydimethylsiloxane (PDMS) membrane at its bottom and (2) embedding hydrogel beads in the spheroids. Culturing spheroids on PDMS increased cell growth and affected glucose/lactate metabolism and CYP3A4 mRNA expression and subsequent enzyme activity. The spheroids comprised 5000 Hep G2 cells and 5000 20 µm-diameter hydrogel beads did not develop a necrotic core for 9 days when cultured on a gas-permeable sheet. In contrast, central necrosis in spheroids lacking hydrogel beads was observed after day 3 of culture, even when using PDMS. These results indicate that the combination of gas-permeable culture equipment and embedded hydrogel beads improves culture 3D spheroids produced from primary or PDX-expanded tumor cells.

Culture systems for three-dimensional tissues, such as multicellular spheroids, are indispensable for high-throughput screening of primary or patient-derived xenograft (PDX)-expanded cancer tissues. Oxygen supply to the center of such spheroids is particularly critical for maintaining cellular functions as well as avoiding the development of a necrotic core. In this study, we evaluated two methods to enhance oxygen supply: (1) using a culture plate with a gas-permeable polydimethylsiloxane (PDMS) membrane on the bottom, and, (2) embedding hydrogel beads in the spheroids. Culturing spheroids on PDMS increased cell growth and affected glucose/lactate metabolism and CYP3A4 mRNA expression and subsequent enzyme activity. The spheroids, comprised of 5000 Hep G2 cells and 5000 20 &micro, m-diameter hydrogel beads, did not develop a necrotic core for nine days when cultured on a gas-permeable sheet. In contrast, central necrosis in spheroids lacking hydrogel beads was observed after day 3 of culture, even when using PDMS. These results indicate that the combination of gas-permeable culture equipment and embedded hydrogel beads improves culture 3D spheroids produced from primary or PDX-expanded tumor cells.

Structural plasticity of dendritic spines, which underlies higher brain functions including learning and memory, is dynamically regulated by the actin cytoskeleton and its associated proteins. Drebrin A is an F-actin-binding protein preferentially expressed in the brain and localized in the dendritic spines of mature neurons. Isoform conversion from drebrin E to drebrin A and accumulation of the latter in dendritic spines occurs during synapse maturation. We have previously demonstrated that drebrin A plays a pivotal role in spine morphogenesis and plasticity. However, it is unclear whether drebrin A plays a specific role in processes required for structural plasticity, and whether drebrin E can substitute in this role. To answer these questions, we analyzed mutant mice (named DAKO mice), in which isoform conversion from drebrin E to drebrin A is disrupted. In DAKO mouse brain, drebrin E continues to be expressed throughout life instead of drebrin A. Electrophysiological studies using hippocampal slices revealed that long-term potentiation of CA1 synapses was impaired in adult DAKO mice, but not in adolescents. In parallel with this age-dependent impairment, DAKO mice exhibited impaired hippocampus-dependent fear learning in an age-dependent manner; the impairment was evident in adult mice, but not in adolescents. In addition, histological investigation revealed that the spine length of the apical dendrite of CA1 pyramidal cells was significantly longer in adult DAKO mice than in wild-type mice. Our data indicate that the roles of drebrin E and drebrin A in brain function are different from each other, that the isoform conversion of drebrin is critical, and that drebrin A is indispensable for normal synaptic plasticity and hippocampus-dependent fear memory in the adult brain.

Introduction Three-dimensional (3D) multicellular spheroids are useful tools for simulation of cellular functions in vitro. However, it is difficult to culture certain epithelial cell types under 3D spheroid conditions because these cells cannot resist autonomous cell death, triggered by disordered cell polarity. The objective of this study was to find a method that enables spheroid culture of such epithelial cells utilizing hydrogel beads without cell death. Methods We used murine E14.5 fetal hepatic cells for the spheroid composition because they are sensitive to disorganized structures. Spheroids were formed by injecting 1-μl fresh medium containing 1000 fetal hepatic cells and the same number of alginate hydrogel beads (20 μm in diameter) into a 3% methylcellulose medium in the presence of dexamethasone and oncostatin M to induce hepatic differentiation. After 7 days of culture, microstructures were observed using hematoxylin and eosin staining and immunostaining using anti-CK8/18 antibody. Albumin secretion rate was determined by the enzyme-linked immunosorbent assay method. In addition, polarity-related proteins, E-cadherin, ezrin, and MRP2 were observed with immunostaining. Results Control spheroids without the use of alginate hydrogel beads showed extensive internal lack of epithelial hepatic cells. The spheroids containing alginate hydrogel beads exhibited sheet- or cord-like structures of epithelial hepatic cells, and it was clear that cell death of epithelial cells had been prevented. Albumin secretion data also supported the improvement of epithelial hepatic cell viability when alginate hydrogel beads were used. Localization of polarity-related proteins indicated the partial reconstitution of cell polarity in the spheroids using alginate hydrogel beads. Conclusion Based on these data, we concluded that the application of alginate hydrogel beads was effective in improving the epithelial hepatic cell culture of multicellular spheroids., Highlights • It was difficult to maintain epithelial fetal hepatic cells in their multicellular spheroids. • The difficulty to maintain the fetal hepatic cells was overcome by embedding of alginate hydrogel beads into the spheroids. • Albumin secretion was improved by embedding of the alginate hydrogel beads. • Cell polarity was partially established in the hybrid spheroids.

The regulation of Sertoli cells by some hormones and signaling factors is important for normal spermatogenesis. Notch signaling is considered to be necessary for normal spermatogenesis in mouse. In this study, we revealed two new facts about Sertoli cells by western blotting experiments on different types of primary cells and microdissected tubules. The first is that Sertoli cells express the Jagged1 ligand in mice testes. The second is that the expression level of Jagged1 oscillates in the seminiferous epithelial cycle. Therefore, we inferred that Jagged1 in Sertoli cells contributes to the Notch signaling involved in spermatogenesis. Furthermore, we examined the regulation of Jagged1 expression and found that Jagged1 expression was suppressed by cAMP signaling and was promoted by TNF-α signaling in Sertoli cells. When cAMP and TNF-α were simultaneously added to Sertoli cells, Jagged1 expression was suppressed. Therefore, cAMP signaling dominates Jagged1 expression over TNF-α signaling. These results suggest that cAMP signaling may cause the periodicity of Jagged1 expression in the seminiferous epithelial cycle, and controlling Jagged1 expression by adding TNF-α or cAMP may contribute to normal spermatogenesis in vitro., Highlights • Jagged1 was expressed in Sertoli cells in mouse testes. • The expression of Jagged1 oscillated in the seminiferous epithelial cycle. • The expression of Jagged1 in Sertoli cells was upregulated by TNF-α and downregulated by cAMP.

A three-dimensional (3D) bone marrow (BM) culture system may facilitate research into the molecular mechanisms involved in hematopoiesis and BM diseases. However, because >90% of BM cells are composed of non-adherent blood cells, it is difficult to organize the dispersed BM cells into 3D multicellular spheroids using conventional aggregation methods such as hanging drop, and rotary shaking culture. The objective of this study was to reproduce BM-like tissue. We reported successful formation of BM aggregates using a 3% methylcellulose (MC) medium. This medium could aggregate even non-adherent materials. In MC medium, BM cells formed tissue-like aggregates within 24 h. Although the cell density of the BM-like tissue is slightly low, sections of the organoids resembled those of intact BM tissue. Cells of the BM-like tissue were approximately 70% viable after 7 days in culture. Staining for CD68, PDGFRα, and CXCL12 indicated that the BM-like tissue contained macrophages, and mesenchymal cells including CXCL12-abundant reticular cells. These results indicated that the method using MC medium effectively reconstitutes the BM-like tissue.

We have previously reported that multi-cellular heteroaggregates comprising murine pancreatic α (αTC1.6) and β (MIN6-m9) cell lines spontaneously acquired islet-like architecture and displayed higher insulin secretion rates. However, the mechanisms of self-organization remain unclear. The objective of this study is to examine the possibility that a sugar chain participates in the mutual recognition of the cells during reconstitution of the islet-like structure in vitro. Using a lectin-binding assay, we identified Erythrina cristagalli agglutinin (ECA), which particularly recognizes the β-galactoside structure on the surfaces of MIN6-m9 cells. The self-organization of αTC1.6 and MIN6-m9 was obstructed using ECA-bound MIN6-m9 cells. Lactose neutralized the ECA's inhibitory effect on the autonomous rearrangement of αTC1.6 and MIN6-m9 cells, indicating that the inhibition of cell arrangement by ECA was mediated via β-galactoside. We concluded that a β-galactoside sugar chain was central to the reconstitution of the pancreatic islet-like architecture in vitro., Highlights • Erythrina cristagalli agglutinin (ECA) lectin bound to the surfaces of MIN6-m9 cells. • ECA-binding had little toxicity on MIN6-m9 cells. • ECA-bound MIN6-m9 cells prevented self-organization of islet-like tissues.

T-box transcription factors play important roles in vertebrate mesoderm formation. Eomesodermin is involved in the initial step of the prospective mesodermal cells recruited near the primitive streak. Then T or Brachyury gene is responsible for general and axial mesodermal development. Tbx6, on the other hand, promotes paraxial mesodermal development while suppressing neural differentiation. Here, we studied differentiative properties of mouse ES cells (mESCs) with its Tbx6 expression regulated under the Tet-off system. mESCs were treated with noggin to promote neural differentiation. When Tbx6 was simultaneously turned on, later neural differentiation of these cells hardly occurred. Next, mESCs were subjected to formation of the embryoid bodies (EBs). When Tbx6 was turned on during EB formation, the rate of later cardiac troponin T (cTnT)-positive cells increased. If the cells were further treated with a wnt inhibitor KY02111 after EB formation, a synergistic increase of cTnT-positive cells occurred. Tbx6 expression in mESCs influenced the constituent ratio of the cardiac myosin light chain types, such that atrial species markedly increased over ventricular ones. These results are coincident with the function of Tbx6 in normal development, in that Tbx6 strongly suppressed neural differentiation while promoting cardiac development in a cooperative manner with wnt inhibition., Highlights • Tbx6 expression in mouse ES cells (mESCs) inhibited neural differentiation. • Tbx6 expression in mESCs increased cardiac muscle synergistically with wnt inhibitor. • Tbx6 expression increased atrial myosin light chains over ventricular chains.

The lack of transplantable pancreatic islets is a serious problem that affects the treatment of patients with type 1 diabetes mellitus. Beta cells can be induced from various sources of stem or progenitor cells, including induced pluripotent stem cells in the near future; however, the reconstitution of islets from β cells in culture dishes is challenging. The generation of highly functional islets may require three-dimensional spherical cultures that resemble intact islets. This review discusses recent advances in the reconstitution of islets. Several factors affect the reconstitution of pseudoislets with higher functions, such as architectural similarity, cell-to-cell contact, and the production method. The actual transplantation of naked or encapsulated pseudoislets and islet-like cell clusters from various stem cell sources is also discussed. Advancing our understanding of the methods used to reconstitute pseudoislets should expand the range of potential strategies available for developing de novo islets for therapeutic applications.

Our objective is to control the reconstitution of liverlike tissues at extrahepatic sites using hepatic progenitor cells (HPCs) andin vitropreculture prior to transplantation. We prepared cell-based hybrid grafts by culturing HPCs isolated from fetal E14.5 mouse livers on biodegradable, highly porous 3-dimensional poly-L-lactic acid (PLLA) scaffolds for 1 week in basal medium (the basal condition) or 10 mM nicotinamide (NA) and 1% dimethyl sulfoxide (DMSO) supplemented conditions (the ND-positive condition) prior to implantation. Sections of hybrid grafts cultured for 1 week showed that HPCs grew and spread on the surface of scaffolds under both basal and ND (+) conditions. Most of these cells were albumin (+) and CK18 (+). CK19 (+) cells were also present under the basal condition but not the ND (+) condition. Cultured hybrid grafts were implanted into the mesenteric leaves of mice and removed after 1 month. Transplanted tissues cultured under the basal condition consisted of albumin (+) hepatocyte-like and CK19 (+) biliary epithelial cell (BEC)-like cells organized in duct-like structures. In contrast, integrated tissues cultured under the ND (+) condition alone had differentiated albumin (+) hepatocyte-like cells and were relatively larger than those under the basal condition. Hepatocyte-like cells of transplanted hybrid grafts cultured under both conditions were periodic acid-Schiff (PAS) staining-positive and expressed transcription factors, hepatocyte nuclear factor (HNF) 4 and CCAAT/enhancer-binding protein (C/EBP) α. These findings suggest that combining progenitor cells andin vitropreculture may potentially regulate liverlike tissues at extrahepatic sites.

Cranial X irradiation can severely impair higher brain function, resulting in neurocognitive deficits. Radiation-induced brain injury is characterized by acute, early and late delayed changes, and morbidity is evident more than 6 months after irradiation. While the acute effects of radiation exposure on the brain are known, the underlying mechanisms remain unclear. In this study, we examined the acute effect of X radiation on synaptic function using behavioral analysis and immunohistochemistry. We found that 10 Gy whole-brain irradiation immediately after conditioning (within 30 min) impaired the formation of fear memory, whereas irradiation 24 h prior to conditioning did not. To investigate the mechanisms underlying these behavioral changes, we irradiated one hemisphere of the brain and analyzed synaptic function and adult neurogenesis immunohistochemically. We focused on drebrin, whose loss from dendritic spines is a surrogate marker of synaptopathy. The intensity of drebrin immunoreactivity started to decrease in the irradiated hemisphere 2 h after exposure. The immunostaining intensity recovered to preirradiation levels by 24 h, indicating that X radiation induced transient synaptic dysfunction. Interestingly, the number of newly generated neurons was not changed at 2 h postirradiation, whereas it was significantly decreased at 8 and 24 h postirradiation. Because irradiation 24 h prior to conditioning had no effect on fear memory, our findings suggest that radiation-induced death of newly-generated neurons does not substantially impact fear memory formation. The radiation-induced synaptic dysfunction likely caused a transient memory deficit during the critical period for fear memory formation (approximately 1-3 h after conditioning), which coincides with a change in drebrin immunostaining in the hippocampus, a structure critical for fear memory formation.

The solute carrier 27A (SLC27A) gene family encodes fatty acid transport proteins (FATPs) and includes 6 members. During fetal and postnatal periods of development, the growing brain requires a reliable supply of fatty acids. Because autism spectrum disorders (ASD) are now recognized as disorders caused by impaired early brain development, it is possible that functional abnormalities of SLC27A genes may contribute to the pathogenesis of ASD. Here, we confirmed the expression of SLC27A3 and SLC27A4 in human neural stem cells derived from human induced pluripotent stem cells, which suggested their involvement in the developmental stage of the central nervous system. Additionally, we resequenced the SLC27A3 and SLC27A4 genes using 267 ASD patient and 1140 control samples and detected 47 (44 novel and 29 nonsynonymous) and 30 (17 novel and 14 nonsynonymous) variants for the SLC27A3 and SLC27A4, respectively, revealing that they are highly polymorphic with multiple rare variants. The SLC27A4 Ser209 allele was more frequently represented in ASD samples. Furthermore, we showed that a SLC27A4 Ser209 mutant resulted in significantly higher fluorescently-labeled fatty acid uptake into bEnd3 cells, a mouse brain capillary-derived endothelial cell line, compared with SLC27A4 Gly209, suggesting that the functional change may contribute to ASD pathophysiology.

MytiLec, a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis), shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling, caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

A three-dimensional (3D) bone marrow (BM) culture system may facilitate research into the molecular mechanisms involved in hematopoiesis and BM diseases. However, because >90% of BM cells are composed of non-adherent blood cells, it is difficult to organize the dispersed BM cells into 3D multicellular spheroids using conventional aggregation methods such as hanging drop, and rotary shaking culture. The objective of this study was to reproduce BM-like tissue. We reported successful formation of BM aggregates using a 3% methylcellulose (MC) medium. This medium could aggregate even non-adherent materials. In MC medium, BM cells formed tissue-like aggregates within 24 h. Although the cell density of the BM-like tissue is slightly low, sections of the organoids resembled those of intact BM tissue. Cells of the BM-like tissue were approximately 70% viable after 7 days in culture. Staining for CD68, PDGFRα, and CXCL12 indicated that the BM-like tissue contained macrophages, and mesenchymal cells including CXCL12-abundant reticular cells. These results indicated that the method using MC medium effectively reconstitutes the BM-like tissue., Highlights • Dispersed-state BM cells can aggregate and organize in MC medium. • BM-like tissue was removed from MC medium after culturing 24 h. • BM-like tissue included macrophages, and PDGFRα+CXCL12+ cells.

The solute carrier 27A (SLC27A) gene family encodes fatty acid transport proteins (FATPs) and includes 6 members. During fetal and postnatal periods of development, the growing brain requires a reliable supply of fatty acids. Because autism spectrum disorders (ASD) are now recognized as disorders caused by impaired early brain development, it is possible that functional abnormalities of SLC27A genes may contribute to the pathogenesis of ASD. Here, we confirmed the expression of SLC27A3 and SLC27A4 in human neural stem cells derived from human induced pluripotent stem cells, which suggested their involvement in the developmental stage of the central nervous system. Additionally, we resequenced the SLC27A3 and SLC27A4 genes using 267 ASD patient and 1140 control samples and detected 47 (44 novel and 29 nonsynonymous) and 30 (17 novel and 14 nonsynonymous) variants for the SLC27A3 and SLC27A4, respectively, revealing that they are highly polymorphic with multiple rare variants. The SLC27A4 Ser209 allele was more frequently represented in ASD samples. Furthermore, we showed that a SLC27A4 Ser209 mutant resulted in significantly higher fluorescently-labeled fatty acid uptake into bEnd3 cells, a mouse brain capillary-derived endothelial cell line, compared with SLC27A4 Gly209, suggesting that the functional change may contribute to ASD pathophysiology.

Tauopathies differ in terms of the brain regions that are affected. In Alzheimer's disease, basal forebrain and hippocampus are mainly involved, while frontotemporal lobar degeneration affects the frontal and temporal lobes and subcortical nuclei including striatum. Over 90% of human cases of tauopathies are sporadic, although the majority of established tau-transgenic mice have had mutations. This prompted us to establish transgenic mice expressing wild-type human tau (Tg601). Old (>14 months old) Tg601 mice displayed decreased anxiety in the elevated plus maze test and impaired place learning in the Morris water maze test. Immunoblotting of brain tissue identified that soluble tau multimer was increased with aging even though insoluble tau was not observed. In the striatum of old Tg601, the level of AT8- or AT180-positive tau was decreased compared with that of other regions, while PHF-1-positive tau levels remained equal. Phosphorylated tau-positive axonal dilations were present mainly in layers V and VI of the prefrontal cortex. Loss of synaptic dendritic spine and decreased immunohistochemical level of synaptic markers were observed in the nucleus accumbens. In vivo 2-[(18)F]fluoro-2-deoxy-d-glucose positron emission tomography analysis also showed decreased activity exclusively in the nucleus accumbens of living Tg601 mice. In Tg601 mice, the axonal transport defect in the prefrontal cortex-nucleus accumbens pathway may lead to decreased anxiety behavior. Differential distribution of hyperphosphorylated tau may cause region-specific neurodegeneration.

Effectively organizing isolated cells to tissue elements having an appropriate microstructure is a fundamental issue in future tissue engineering, but biological cell-to-cell adhesion is too weak to assemble single cells directly. In order to overcome the difficulty, we applied an Avidin-Biotin Binding System (ABBS) to cell surfaces, and avidinylated and biotinylated cells could mutually bind in the short time they were mixed together. Unlike conventional intact cells, ABBS helped make larger spheroids. Interestingly, avidinylated and biotinylated cell adherence occurred within 1 sec using laser trapping, enabling single cell manipulation. We showed precise, direct single-cell-based tissue assembly using ABBS and optical tweezers, followed by damage-free tissue culture. The combination of ABBS and single cell manipulation has considerable potential for use in application such as tissue engineering, regenerative medicine, and drug screening system.

We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.

C/EBPalpha is a key transcription factor indispensable for the onset of gluconeogenesis in perinatal liver. However, C/EBPalpha was already expressed in fetal liver, suggesting that the expression of C/EBPalpha alone does not account for the dramatic increase of the expression of metabolic genes, and hence an additional factor(s) is expected to function cooperatively with C/EBPalpha in perinatal liver. We show here that expression of Foxo1 was sharply increased in the perinatal liver and augmented C/EBPalpha-dependent transcription. Foxo1 bound C/EBPalpha via its forkhead domain, and Foxo1 bound to the promoter of a gluconeogenic gene, phosphoenolpyruvate carboxykinase (PEPCK), in a C/EBPalpha-dependent manner in vivo. Insulin inhibited the expression of PEPCK in a culture of fetal liver cells, and also the C/EBPalpha-dependent transcription enhanced by Foxo1. These results indicate that Foxo1 regulates gluconeogenesis cooperatively with C/EBPalpha, and also links insulin signaling to C/EBPalpha during liver development.

Efficient cell attachment to biodegradable polymer scaffolds is a necessary prerequisite in tissue engineering. However, it is difficult to evenly cover scaffold surfaces with cells because scaffolds are generally highly porous, with complex three-dimensional (3D) surfaces. In this article, we demonstrate the efficiency of avidin-biotin binding systems (ABBS) for the initial attachment of biotinylated Hep G2 cells to avidin adsorbed flat, two-dimensional (2D) and highly porous 3D poly L-lactic acid (PLLA) surfaces. The potential toxicity of biotinylation and/or strong ABBS binding forces was also investigated. ABBS assisted Hep G2 cells to adhere to a flat PLLA surface within 10min; the proliferation of these attached cells was comparable with control intact cells cultured on collagen. Hepatic functions of the attached cells, such as albumin secretion, induction of CYP1A1 and CYP1A2 genes, and metabolic capacity of CYP1A1/2 as measured by the ethoxyresorufin O-deethylase assay, were not significantly changed. Also, a stimulus of a cytokine: oncostatin M (OSM) phosphorylated an intracellular signaling molecule, extracellular signal-related kinase 1 (ERK1) via transmembrane receptor complex, at 24h after inoculation by ABBS. In addition, efficient attachment of Hep G2 cells to a highly porous PLLA 3D scaffold was demonstrated. These results clearly show that ABBS is useful for rapidly trapping cells in both biodegradable, polymer-based, flat 2D surfaces, and in highly porous 3D scaffolds. Furthermore, binding hepatic cells by this technique has only small effects on liver-specific functions, or on signal transfer ability of transmembrane receptor complexes.

Cell surface molecules are not only important for cell–cell interactions but also useful for a marker to define cell types and differentiation stages. Unlike hematopoietic system in which numerous such antigens have been identified, only a few cell surface molecules have been used to define differentiation stage of hepatocytes. In order to identify such cell surface molecules, we performed DNA microarray analysis using mRNA from fetal hepatocytes in E12.5 and E17.5 mice and cDNAs encoding a membrane protein were selected. Northern blot analysis was employed to confirm the genes upregulated during maturation of fetal hepatocytes and neuritin, a GPI-anchored protein, was found as a membrane protein expressed in hepatocytes, but not in nonparenchymal cells. Its expression increased along with liver development and the maximum expression was achieved from the neonatal to adult stage. The neuritin protein was localized in sinusoidal lumen of hepatocytes in adult liver. Partial hepatectomy transiently downregulated the expression of neuritin. The expression of neuritin mRNA in C/EBPα deficient liver was reduced to about 50% of that of wild type mice. Thus, neuritin expression is well correlated to the maturation of hepatocytes and can be a useful tool to define the differentiation stage of hepatocytes.

Although cells isolated from fetal liver are one of the major sources for liver tissue engineering, it is still very difficult to induce them to fully differentiate in vitro into mature hepatocytes. We therefore investigated the effects of nicotinamide (NA), dimethyl sulfoxide (DMSO), and oncostatin M (OSM) on differentiation in terms of the expression of various liver-specific functions, because these factors have been reported to induce the emergence of possible hepatocyte progenitor cells (small hepatocytes) in adult rat hepatocyte culture or maturation of fetal mouse liver cells in culture. Fetal liver cells isolated from mouse embryos were cultured for 5 weeks in collagen-precoated plates. NA (10 mM) and DMSO (1%) remarkably enhanced the emergence of small hepatocytes, and OSM also synergistically enhanced the selective growth of small hepatocytes and inhibited the growth of blood cell populations. In the presence of these three factors, such small hepatocytes became dominant in culture, so that they covered almost 60–70% of confluence after week 2. In addition, some of them piled up over the small hepatocyte monolayer and displayed distinctively differentiated morphology, such as the emergence of binucleated cells, formation of tight gap junctions, and possible bile duct structures. Although OSM alone had very weak effects on hepatocyte functions, albumin secretion and cytochrome P450IA1/2 capacity were greatly enhanced when combined with NA or DMSO. This functional observation closely agreed with the emergence of small hepatocytes. In contrast, ammonium removal was strongly dependent on DMSO alone. DNA amount basis functions of fetal cells with three factors at week 5 were 1/7 for albumin secretion, 3 times higher for ammonium removal, and 1/10 for P450 capacity, compared with those of cultured adult mouse hepatocytes. These results show that inclusion of NA, DMSO, and OSM in the culture medium significantly enhances in vitro maturation of fetal liver cells when compared with conventional culture conditions.

The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca(2+) influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.

Ammonia metabolism of ratprimary hepatocytes and a human hepatocyte cell line,Huh 7, at different concentrations of glutamine,glucose and ammonia was examined. During theincubation of the primary hepatocyte cells, glutamineand ammonia concentrations decreased, that of ureaincreased, and that of glucose remained the same. Inthe case of Huh 7 cells, glucose was consumed rapidly,the concentration of ammonia increased and that of urearemained the same. The major energy sources amongmedium components were glutamine for the primary cellsand glucose for Huh 7 cells, although the primaryhepatocytes may utilize intracellular glycogen asenergy source. As the glutamine concentration in theincubation medium increased, the specific rates of notonly glutamine consumption, but also ammonia productionby the primary cells and Huh 7 cells increased. Besides, specific urea production rate by the primarycells increased then. Increase of glucoseconcentration had no effect on glutamine and ammoniametabolism by both cells, although it increased glucoseconsumption by Huh 7 cells. The incubation of theprimary cells with higher ammonia concentrationincreased all specific rates of glutamine consumption,ammonia consumption and urea production. An increasein the ammonia concentration to 5 mM changed theammonia metabolism from production to consumption andincreased the specific glucose consumption rate. Consequently, increases in the glutamine and ammoniaconcentrations were revealed to have negative andpositive effects, respectively, on decreasing ammoniaconcentration by both of rat primary hepatocytes andHuh 7 cells.

Earlier work has suggested that Fyn tyrosine kinase plays an important role in synaptic plasticity. To understand the downstream targets of Fyn signaling cascade in neurons, we generated transgenic mice expressing either a constitutively activated form of Fyn or native Fyn in neurons of the forebrain. Transgenic mice expressing mutant Fyn exhibited higher seizure activity and were prone to sudden death. Mice overexpressing native Fyn did not show such an obvious epileptic phenotype, but they exhibited accelerated kindling in response to once-daily stimulation of the amygdala. Tyrosine phosphorylation of at least three proteins was enhanced in the forebrains of both native and mutant fyn transgenic mice; tyrosine phosphorylation of these three proteins was reduced infyn knockout mice, suggesting that they are substrates of Fyn. One of these proteins was identified as the subunit 2B (NR2B) of theN-methyl-d-aspartate (NMDA) receptor. Administration of MK-801, a noncompetitive NMDA receptor antagonist, retarded kindling in mice overexpressing native Fyn, as well as wild-type mice, suggests that the accelerated kindling in mice overexpressing Fyn is also mediated by the NMDA receptor activity. Our results thus suggest that tyrosine phosphorylation by Fyn might be involved in regulation of the susceptibility of kindling, one form of the NMDA receptor-mediated neuronal plasticity.

To examine the physiological role of the Fyn tyrosine kinase in neurons, we generated transgenic mice that expressed a fyn cDNA under the control of the calcium/calmodulin-dependent protein kinase IIα promoter. With this promoter, we detected only low expression of Fyn in the neonatal brain. In contrast, there was strong expression of the fyn-transgene in neurons of the adult forebrain. To determine whether the impairment of long-term potentiation (LTP) observed in adult fyn-deficient mice was caused directly by the lack of Fyn in adult hippocampal neurons or indirectly by an impairment in neuronal development, we generated fyn-rescue mice by introducing the wild-type fyn-transgene into mice carrying a targeted deletion in the endogenous fyn gene. In fyn-rescue mice, Schaffer collateral LTP was restored, even though the morphological abnormalities characteristic of fyn-deficient mice were still present. These results suggest that Fyn contributes, at least in part, to the molecular mechanisms of LTP induction.

We report a method for the rapid production of cellular aggregates without electric power and cell modification. We focused on the swelling property of a solution containing a high molecular material, methylcellulose (MC), which immediately absorbs a small amount of solvent and fills the space occupied by the solvent. When 1 μl of a suspension of 1000 animal cells in normal culture medium was injected into the 3% MC medium, the normal medium was rapidly absorbed by the surrounding MC medium. Suspended cells were simultaneously trapped on the interfaces between the normal and MC media; they were finally pulled together and held in the MC medium. This event was nearly complete within the first 10 min. Moreover, MC medium-dependent aggregation was observed when polystyrene microspheres of different sizes (diameter, 100 nm-100 μm) were added. Furthermore, we demonstrated the stepwise fabrication of multi-layered aggregates with embedded structures. These methods for creating engineered aggregates should enhance the study of three-dimensional cultures comprising two or more cell types with well-designed structures.

Multicellular heterospheroids including two or more cell types have some tissue/organ properties and can be used in cell-to-cell interaction studies. However, the spheroid formation is difficult to control because the adhesion efficacy is different in each cell type. To solve this, we applied a rapid cell-to-cell adhesion method, avidin–biotin (AB) binding, to spheroid formation. Introduction of avidin or biotin molecules to the cell surfaces of Mile Sven 1 (MS1) cells promoted formation of spheroid in minutes. This method allowed the construction of heterospheroids having homogenous distributions of different cell types. Interestingly, cells showed self-organization and MS1 cells formed networks with Hep G2 cells. NIH3T3 cells also remodeled when mixed with Hep G2 cells. In contrast, a combination of MS1 and NIH3T3 cells failed to show pattern formation, indicating that self-organization was based on the composition of cell types. Actin polymerization not cell proliferation was the dominant factor in remodeling of heterospheroids in the first 24 h. We also demonstrated the self-organization of spheroids comprising three different cell types. The new technology to assemble cells is important not only to study cell-to-cell interaction but also to make three-dimensional complicated tissues.

Homeostatic synaptic plasticity (HSP) is important for maintaining neurons' excitability within the dynamic range and for protecting neurons from unconstrained long-term potentiation that can cause breakdown of synapse specificity (Turrigiano [2008] Cell 135:422-435). Knowledge of the molecular mechanism underlying this phenomenon remains incomplete, especially for the rapid form of HSP. To test whether HSP in adulthood depends on an F-actin binding protein, drebrin A, mice deleted of the adult isoform of drebrin (DAKO) but retaining the embryonic isoform (drebrin E) were generated. HSP was assayed by determining whether the NR2A subunit of N-methyl-D-aspartate receptors (NMDARs) can rise rapidly within spines following the application of an NMDAR antagonist, D-APV, onto the cortical surface. Electron microscopic immunocytochemistry revealed that, as expected, the D-APV treatment of wild-type (WT) mouse cortex increased the proportion of NR2A-immunolabeled spines within 30 minutes relative to basal levels in hemispheres treated with an inactive enantiomer, L-APV. This difference was significant at the postsynaptic membrane and postsynaptic density (i.e., synaptic junction) as well as at nonsynaptic sites within spines and was not accompanied by spine size changes. In contrast, the D-APV treatment of DAKO brains did not augment NR2A labeling within the spine cytoplasm or at the synaptic junction, even though basal levels of NR2A were not significantly different from those of WT cortices. These findings indicate that drebrin A is required for the rapid (