記述言語：日本語   出版者・発行元：(一社)日本脳循環代謝学会  

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記述言語：日本語   出版者・発行元：(一社)日本脳循環代謝学会  

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記述言語：日本語   出版者・発行元：(一社)日本脳循環代謝学会  

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Replication competent oncolytic herpes simplex virus (HSV) vectors have been used extensively to treat solid tumors with promising results. However, highly defective HSV vectors will be needed for applications that require sustained therapeutic gene expression in the absence of vector-related toxicity or inflammation. These vectors require complementing cell lines for their manufacture, creating significant challenges to achieve high yields of infectious virus particles. We recently described an improved upstream process for the production of a non-cytotoxic HSV vector for gene therapy applications. Here, we sought to optimize the downstream conditions for purification and long-term storage of the same vector, JΔNI5. We compared different methods to remove cellular impurities and concentrate the vector by monitoring both physical and biological titers, resulting in the establishment of optimal conditions for vector production. To optimize the long-term storage parameters for non-cytotoxic HSV vectors, we evaluated vector stability at low temperature and sensitivity to freeze-thaw cycles. We report that suboptimal purification and storage methods resulted in loss of vector viability. Our results describe effective and reproducible protocols for purification and storage of HSV vectors for pre-clinical studies.

DOI： 10.1016/j.omtm.2022.06.007

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Retinitis pigmentosa (RP) is a genetically heterogeneous group of inherited retinal disorders involving the progressive dysfunction of photoreceptors and the retinal pigment epithelium, for which there is currently no treatment. The rd6 mouse is a natural model of autosomal recessive retinal degeneration. Given the known contributions of oxidative stress caused by reactive oxygen species (ROS) and selective inhibition of potent ROS peroxynitrite and OH·by H2 gas we have previously demonstrated, we hypothesized that ingestion of H2 water may delay the progression of photoreceptor death in rd6 mice. H2 mice showed significantly higher retinal thickness as compared to controls on optical coherence tomography. Histopathological and morphometric analyses revealed higher thickness of the outer nuclear layer for H2 mice than controls, as well as higher counts of opsin red/green-positive cells. RNA sequencing (RNA-seq) analysis of differentially expressed genes in the H2 group versus control group revealed 1996 genes with significantly different expressions. Gene and pathway ontology analysis showed substantial upregulation of genes responsible for phototransduction in H2 mice. Our results show that drinking water high in H2 (1.2-1.6 ppm) had neuroprotective effects and inhibited photoreceptor death in mice, and suggest the potential of H2 for the treatment of RP.

DOI： 10.1038/s41598-022-17903-8

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The application of recombinant adeno-associated viruses (rAAVs) for gene therapy faces certain challenges, including genome packaging of non-vector sequences. Inverted terminal repeats (ITRs) flanking the rAAV genome, comprising three inverted repeat regions (A, B, and C) and a non-inverted repeat region (D), contribute to non-vector genome packaging. We aimed to circumvent this issue by comparing the properties of rAAV containing DNA plasmids and PCR-amplified transgenes, including a single copy of the AD sequence (rAAV-pAD/L-AD, respectively), which is a truncated form of ITR, with those of wild-type ITR genome (single-stranded and self-complementary AAV; ssAAV and scAAV). The packaging efficiency of rAAV-pAD/L-AD was found to be comparable to that of scAAV, whereas the transduction efficiency of rAAV-pAD/L-AD was lower than that of ss/scAAV. Remarkably, rAAV-L-AD reduced the plasmid backbone packaging contamination compared to ss/scAAV. Furthermore, to confirm the functionality of this system, we generated a rAAV-L-AD harboring a short hairpin RNA targeting ATP5B (rAAV-L-AD-shATP5B) and found that it caused a significant decrease in ATP5B mRNA levels when transduced into HEK293EB cells, suggesting that it was functional. Thus, our system successfully packaged L-AD into capsids with minimal contamination of plasmid DNA, offering a novel functional packaging platform without causing plasmid backbone encapsidation.

DOI： 10.1038/s41434-021-00299-x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Oncolytic herpes simplex viruses (oHSV) are under development for the treatment of a variety of human cancers, including breast cancer, a leading cause of cancer mortality among women worldwide. Here we report the design of a fully retargeted oHSV for preferential infection of breast cancer cells through virus recognition of GFRα1, the cellular receptor for glial cell-derived neurotrophic factor (GDNF). GFRα1 displays a limited expression profile in normal adult tissue, but is upregulated in a subset of breast cancers. We generated a recombinant HSV expressing a completely retargeted glycoprotein D (gD), the viral attachment/entry protein, that incorporates pre-pro-GDNF in place of the signal peptide and HVEM binding domain of gD and contains a deletion of amino acid 38 to eliminate nectin-1 binding. We show that GFRα1 is necessary and sufficient for infection by the purified recombinant virus. Moreover, this virus enters and spreads in GFRα1-positive breast cancer cells in vitro and caused tumor regression upon intratumoral injection in vivo. Given the heterogeneity observed between and within individual breast cancers at the molecular level, these results expand our ability to deliver oHSV to specific tumors and suggest opportunities to enhance drug or viral treatments aimed at other receptors.

DOI： 10.3390/ijms21228815

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Non-toxic herpes simplex virus (HSV) vectors can be generated by functional deletion of all immediate-early (IE) genes, providing a benign vehicle with potential for gene therapy. However, deletion of multiple IE genes raises manufacturing concerns and thus limits clinical application of these vectors. To address this issue, we previously developed a novel production cell line, called U2OS-ICP4/27, by lentiviral transduction of human osteosarcoma U2OS cells with two essential HSV IE genes, ICP4 and ICP27. To optimize the process of vector manufacturing on this platform, we evaluated several cell culture parameters of U2OS-ICP4/27 for high-titer and -quality production of non-toxic HSV vectors, revealing that the yields and functionality of these vectors can be significantly influenced by culturing conditions. We also found that several chemical compounds can enhance the replication of non-toxic HSV vectors and their release from producer cells into the supernatants. Notably, the vector produced by our optimized protocol displayed a greatly improved vector yield and quality and showed elevated transgene expression in cultures of primary dorsal root ganglion neurons. Taken together, our optimized production approach emerges as a relevant protocol for high-yield and high-quality preparation of non-toxic HSV-based gene therapy vectors.

DOI： 10.1016/j.omtm.2020.03.014

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1177/1744806920904462

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記述言語：日本語   出版者・発行元：日本医科大学医学会  

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DOI： 10.1016/j.omtm.2018.10.015

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DOI： 10.1128/JVI.00536-18

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DOI： 10.3390/diseases6030074

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

The ability of herpes simplex virus (HSV) to establish lifelong latency in neurons suggests that HSV-derived vectors hold promise for gene delivery to the nervous system. However, vector toxicity and transgene silencing have created significant barriers to vector applications to the brain. Recently, we described a vector defective for all immediate-early gene expression and deleted for the joint region between the two unique genome segments that proved capable of extended transgene expression in non-neuronal cells. Sustained expression required the proximity of boundary elements from the latency locus. As confirmed here, we have also found that a transgene cassette introduced into the ICP4 locus is highly active in neurons but silent in primary fibroblasts. Remarkably, we observed that removal of the virion host shutoff (vhs) gene further improved transgene expression in neurons without inducing expression of viral genes. In rat hippocampus, the vhs-deleted vector showed robust transgene expression exclusively in neurons for at least 1 month without evidence of toxicity or inflammation. This HSV vector design holds promise for gene delivery to the brain, including durable expression of large or complex transgene cassettes.

DOI： 10.1016/j.omtm.2017.06.001

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Previously we reported a new series of highly defective herpes simplex virus type 1 (HSV-1) vectors that were functionally devoid of all viral immediately early (IE) genes, resulting in virtual absence of viral gene expression. Nevertheless, a reporter gene cassette inserted into the vector flanked by boundary elements from the viral latency locus showed high, persistent reporter gene activity in non-neuronal cells while an independent expression cassette inserted into a deleted ICP4 locus remained almost silent. In contrast to non-neuronal cells, we show here that the ICP4 locus cassette permitted robust reporter gene expression in a diversity of neurons following stereotactic injection of different rat brain regions; transgene expression in the hippocampus lasted up to 6 months and was essentially restricted to neurons. No evidence of neuronal cell toxicity or induction of inflammatory cell infiltrates was observed. An independent reporter gene cassette located in an intergenic region remained silent, indicating that the transgene promoter and/or insertion site are critical for sustained expression. These findings suggest the suitability of this vector for therapeutic intervention into diseases of the central nervous system that require the expression of large and/or multiple therapeutic transgenes.

DOI： 10.1038/s41598-017-01635-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate-tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up-regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low-density lipoprotein-related protein 5/6, transgenic mice expressing stable -catenin, and in vitro studies, we show a role of Wnt signaling through -catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a -catenin-independent manner. Conclusion: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both -catenin-dependent and -independent manners. (Hepatology 2016;64:1652-1666)

DOI： 10.1002/hep.28774

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Oncolytic herpes simplex virus (HSV) vectors have attracted increasing attention as novel anti-cancer agents. HSV entry is triggered by the binding of glycoprotein D (gD) to its receptors, such as herpesvirus entry mediator or nectin-1. We have recently reported the construction of a fully retargeted HSV platform that incorporates single-chain antibodies (scFv) into gD to mediate entry exclusively via tumor-associated antigens. In this study, we created an scFv directed against epithelial cell adhesion molecule (EpCAM), a recognized carcinoma-associated antigen, and inserted it into the retargeted HSV platform that is ablated for gD recognition of its canonical receptors and contains the entry-enhancing mutations in gB we previously identified. We observed that both initial entry and subsequent cell-to-cell spread of the retargeted virus were stringently dependent on cellular EpCAM expression. Interestingly, the retargeted virus developed larger plaques on some of the human tumor lines tested than the control virus bearing wild-type gD. Intratumoral injection of the retargeted virus revealed antitumor activity in a mouse xenograft model. These observations illustrate the versatility of our retargeted HSV platform as it allows expansion of the oncolytic virus toolbox for the treatment of diverse cancers.

DOI： 10.1038/gt.2016.17

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記述言語：日本語   出版者・発行元：日本臨床プロテオーム研究会  

DOI： 10.14905/jscp.2016.0_16

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Glycosphingolipids (GSLs) are glycoconjugates that function as mediators of cell adhesion and modulators of signal transduction. Some well-defined markers of undifferentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are glycoconjugates, such as SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. However, Comprehensive GSL profiles of hiPSCs have not yet been elucidated. The global images of GSLs from the parental cells, hiPSCs, and differentiated cells revealed that there are parental cell-independent specific glycolipids, including Globo H (fucosyl-Gb5Cer) and H type1 antigen (fucosyl-Lc4Cer) that are novel markers for undifferentiated hiPSCs. Interestingly, undifferentiated hiPSCs expressed H type 1 antigen, specific for blood type O, regardless of the cells' genotypes. Thus, in this study, we defined the dynamics of GSL remodeling during reprogramming from parental cell sets to iPSC sets and thence to iPSC-neural cells.

DOI： 10.1038/srep14988

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5' to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors.

DOI： 10.1073/pnas.1423556112

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Glioblastoma multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSVs) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals, but their efficacy in early phase patient trials has been limited. Instead of attenuating the virus with mutations in virulence genes, we engineered four copies of the recognition sequence for miR-124 into the 3'UTR of the essential ICP4 gene to protect healthy tissue against lytic virus replication; miR-124 is expressed in neurons but not in glioblastoma cells. Following intracranial inoculation into nude mice, the miR-124-sensitive vector failed to replicate or show overt signs of pathogenesis. To address the concern that this safety feature may reduce oncolytic activity, we inserted the miR-124 response elements into an unattenuated, human receptor (EGFR/ EGFRvIII)-specific HSV vector. We found that miR-124 sensitivity did not cause a loss of treatment efficiency in an orthotopic model of primary human GBM in nude mice. These results demonstrate that engineered miR-124 responsiveness can eliminate off-target replication by unattenuated oHSV without compromising oncolytic activity, thereby providing increased safety.

DOI： 10.1038/mt.2014.177

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes.

DOI： 10.5966/sctm.2013-0179

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE SOC STUDY XENOBIOTICS  

The use of human induced pluripotent stem (iPS) cells would be of great value for a variety of applications involving drug development studies. Several reports have been published on the differentiation of human iPS cells into hepatocyte-like cells; however, the cells were insufficient for application in drug metabolism studies. In this study, we aimed to establish effective methods for differentiation of human iPS cells into hepatocytes. Two human iPS cell lines were differentiated by addition of activin A, dimethyl sulfoxide, hepatocyte growth factor, oncostatin M, and dexamethasone. The differentiated cells expressed hepatocyte markers and drug-metabolizing enzymes, revealing that the human iPS cells were differentiated into hepatocyte-like cells. Expression of CYP3A4 and UGT1A1 mRNAs increased with treatment with typical inducers of the enzymes, and the response of the cells against the inducers was similar to that of human hepatocytes. Furthermore, the drug-metabolizing activity of CYP3A4, as monitored by testosterone 6 beta-hydroxylase activity, was elevated by these inducers. In conclusion, we established methods for differentiation of hepatocyte-like cells expressing drug metabolizing activity from human iPS cells. The hepatocyte-like cells derived from human iPS cells will be useful for drug metabolism studies.

DOI： 10.2133/dmpk.DMPK-13-RG-104

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

Redirecting differentiation of somatic cells by over-expression of transcription factors is a promising approach for regenerative medicine, elucidation of pathogenesis and development of new therapies. We have previously defined a transcription factor combination, that is, CRX, RAX and NEUROD, that can generate photosensitive photoreceptor cells from human iris cells. Here, we show that human dermal fibroblasts are differentiated to photoreceptor cells by the same transcription factor combination as human iris cells. Transduction of a combination of the CRX, RAX and NEUROD genes up-regulated expression of the photoreceptor-specific genes, recoverin, blue opsin and PDE6C, in all three strains of human dermal fibroblasts that were tested. Additional OTX2 gene transduction increased up-regulation of the photoreceptor-specific genes blue opsin, recoverin, S-antigen, CNGB3 and PDE6C. Global gene expression data by microarray analysis further showed that photoreceptor-related functional genes were significantly increased in induced photoreceptor cells. Functional analysis, that is, patch-clamp recordings, clearly revealed that induced photoreceptor cells from fibroblasts responded to light. Both the NRL gene and the NR2E3 gene were endogenously up-regulated in induced photoreceptor cells, implying that exogenous CRX, RAX, OTX2 and NEUROD, but not NRL, are sufficient to generate rod photoreceptor cells.

DOI： 10.1111/gtc.12127

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE SOC STUDY XENOBIOTICS  

Human induced pluripotent stem (iPS) cells were differentiated into the endoderm using activin A and were then treated with fibroblast growth factor 2 (FGF2) for differentiation into intestinal stem cell-like cells. These immature cells were then differentiated into enterocyte-like cells using epidermal growth factor (EGF) in 2% fetal bovine serum (FBS). At the early stage of differentiation, mRNA expression of caudal type homeobox 2 (CDX2), a major transcription factor related to intestinal development and differentiation, and leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), an intestinal stem cell marker, was markedly increased by treatment with FGF2. When cells were cultured in medium containing EGF and a low concentration of FBS, mRNAs of specific markers of intestinal epithelial cells, including sucrase-isomaltase, the intestinal oligopeptide transporter SLC15A1/peptide transporter 1 (PEPT1), and the major metabolizing enzyme CYP3A4, were expressed. In addition, sucrase-isomaltase protein expression and uptake of beta-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (beta-Ala-Lys-AMCA), a fluorescence-labeled substrate of the oligopeptide transporter, were detected. These results demonstrate a simple and direct method for differentiating human iPS cells into functional enterocyte-like cells.

DOI： 10.2133/dmpk.DMPK-13-RG-005

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COMPANY OF BIOLOGISTS LTD  

Embryonic stem (ES) cells recapitulate normal developmental processes and serve as an attractive source for routine access to a large number of cells for research and therapies. We previously reported that ES cells cultured on M15 cells, or a synthesized basement membrane (sBM) substratum, efficiently differentiated into an endodermal fate and subsequently adopted fates of various digestive organs, such as the pancreas and liver. Here, we established a novel hepatic differentiation procedure using the synthetic nanofiber (sNF) as a cell culture scaffold. We first compared endoderm induction and hepatic differentiation between murine ES cells grown on sNF and several other substrata. The functional assays for hepatocytes reveal that the ES cells grown on sNF were directed into hepatic differentiation. To clarify the mechanisms for the promotion of ES cell differentiation in the sNF system, we focused on the function of Rac1, which is a Rho family member protein known to regulate the actin cytoskeleton. We observed the activation of Rac1 in undifferentiated and differentiated ES cells cultured on sNF plates, but not in those cultured on normal plastic plates. We also show that inhibition of Rac1 blocked the potentiating effects of sNF on endoderm and hepatic differentiation throughout the whole differentiation stages. Taken together, our results suggest that morphological changes result in cellular differentiation controlled by Rac1 activation, and that motility is not only the consequence, but is also able to trigger differentiation. In conclusion, we believe that sNF is a promising material that might contribute to tissue engineering and drug delivery.

DOI： 10.1242/jcs.129767

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Glycogen storage disease type Ib (GSDIb) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), which leads to neutrophil dysfunction. However, the underlying causes of these dysfunctions and their relationship with glucose homeostasis are unclear. Induced pluripotent stem cells (iPSCs) hold a great promise for advances in developmental biology, cell-based therapy and modeling of human disease. Here, we examined the use of iPSCs as a model for GSDIb. In this study, one 2-year-old patient was genetically screened and diagnosed with GSDIb. We established iPSCs and differentiated these cells into hepatocytes and neutrophils, which comprise the main pathological components of GSDIb. Cells that differentiated into hepatocytes exhibited characteristic albumin secretion and indocyanine green uptake. Moreover, iPSC-derived cells generated from patients with GSDIb metabolic abnormalities recapitulated key pathological features of the diseases affecting the patients from whom they were derived, such as glycogen, lactate, pyruvate and lipid accumulation. Cells that were differentiated into neutrophils also showed the GSDIb pathology. In addition to the expression of neutrophil markers, we showed increased superoxide anion production, increased annexin V binding and activation of caspase-3 and caspase-9, consistent with the GSDIb patient's neutrophils. These results indicate valuable tools for the analysis of this pathology and the development of future treatments.

DOI： 10.1111/gtc.12101

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記述言語：英語   出版者・発行元：(一社)日本癌学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1186/2042-4280-4-2

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記述言語：日本語   出版者・発行元：(一社)日本癌治療学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BIOMED CENTRAL LTD  

Background: hiPSCs are generated through epigenetic reprogramming of somatic tissue. Genomic imprinting is an epigenetic phenomenon through which monoallelic gene expression is regulated in a parent-of-origin-specific manner. Reprogramming relies on the successful erasure of marks of differentiation while maintaining those required for genomic imprinting. Loss of imprinting (LOI), which occurs in many types of malignant tumors, would hinder the clinical application of hiPSCs.
Results: We examined the imprinting status, expression levels and DNA methylation status of eight imprinted genes in five independently generated hiPSCs. We found a low frequency of LOI in some lines. Where LOI was identified in an early passage cell line, we found that this was maintained through subsequent passages of the cells. Just as normal imprints are maintained in long-term culture, this work suggests that abnormal imprints are also stable in culture.
Conclusions: Analysis of genomic imprints in hiPSCs is a necessary safety step in regenerative medicine, with relevance both to the differentiation potential of these stem cells and also their potential tumorigenic properties.

DOI： 10.1186/1471-2156-14-32

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double-stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome-integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA. © 2012 Institute of Zoology, Chinese Academy of Sciences.

DOI： 10.1111/j.1744-7917.2012.01569.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

Since BCR-ABL plays an essential role in the growth factor-independent proliferation of Philadelphia chromosome (Ph)+leukemia cells, imatinib treatment of Ph+leukemia cells inactivates signaling pathways of BCR-ABL, and subsequent addition of growth factors (GFs) could restore the signaling pathways without reactivating BCR-ABL. Here we demonstrated that non-lymphoid Ph+leukemia cell lines responded to diverse GFs depending on their immunophenotype and gene expression of transcription factors and GF receptors, while lymphoid Ph+leukemia cell lines restrictively responded to flit3 ligand and interleukin-7, suggesting that GF sensitivity of imatinib-treated Ph+leukemia cells could be powerful for specifying their distinctive lineage. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.leukres.2012.10.001

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Human bone morphogenetic protein 4 (BMP4) fused to the collagen-binding domain (CBD) of fbronectin was constructed and expressed in a silkworm-baculovirus expression system. When recombinant BmNPV was injected into the silkworm larvae and harvested after approximately 4 days, 0.22 mg/ml (0.1 mg/larva) of recombinant CBD-BMP4 was secreted into the silkworm haemolymph. Interestingly, cultured silkworm cells infected with the same recombinant virus could not secrete the recombinant CBD-BMP4 into culture media. The purifed rCBD-BMP4 showed Smad- and MAPK-stimulating activities in human UET-13 cells.

DOI： 10.11416/jibs.82.2_039

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The development of a three-dimensional (3D) culture system is very important for regenerative medicine and drug discovery applications of stem cell technology because the 3D culture condition could mimic the stem cell environment in vivo and support accurate differentiation. This chapter describes a novel 3D culture technique for efficient induction of adipogenic differentiation. This 3D culture system provides an easy way to allow cells to form a 3D spheroid structure without any matrix derived from animal and chemical substances. We firstly describe the details of the 3D culture technique using human mesenchymal stem/progenitor cells (MPCs) and its optimization. Then we elaborate on the protocol of efficient induction for adipogenic differentiation and adipocyte-specific gene expression, including peroxisome proliferator-activated receptor-γ, with our culture system in human MPCs. © 2013 Springer Science+Business Media New York.

DOI： 10.1007/978-1-62703-155-4_20

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

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.

DOI： 10.1002/eji.201242530

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：KYUSHU UNIV, FACULTY AGRICULTURAL PUBLICATIONS  

RNA interference (RNAi) triggered by long double-stranded RNA (dsRNA) transcribed from chromosomal DNA has a lot of advantages in high-throughput analyses of gene function. In this report, we have constructed Gatewar (R)-based RNAi induction vectors, by which we efficiently integrated expression cassettes for long hairpin dsRNA into chromosomal DNA of Bombyx mori cells. RNAi induced by the hairpin dsRNAs using our constructs decreased a reporter gene activity by approximately 70-80% in cultured B. mori cells.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Examples of direct differentiation by defined transcription factors have been provided for beta-cells, cardiomyocytes and neurons. In the human visual system, there are four kinds of photoreceptors in the retina. Neural retina and iris-pigmented epithelium (IPE) share a common developmental origin, leading us to test whether human iris cells could differentiate to retinal neurons. We here define the transcription factor combinations that can determine human photoreceptor cell fate. Expression of rhodopsin, blue opsin and green/red opsin in induced photoreceptor cells were dependent on combinations of transcription factors: A combination of CRX and NEUROD induced rhodopsin and blue opsin, but did not induce green opsin; a combination of CRX and RX induced blue opsin and green/red opsin, but did not induce rhodopsin. Phototransduction-related genes as well as opsin genes were up-regulated in those cells. Functional analysis; i.e. patch clamp recordings, clearly revealed that generated photoreceptor cells, induced by CRX, RX and NEUROD, responded to light. The response was an inward current instead of the typical outward current. These data suggest that photosensitive photoreceptor cells can be generated by combinations of transcription factors. The combination of CRX and RX generate immature photoreceptors: and additional NEUROD promotes maturation. These findings contribute substantially to a major advance toward eventual cell-based therapy for retinal degenerative diseases.

DOI： 10.1371/journal.pone.0035611

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MARY ANN LIEBERT INC  

We have established a serum- and feeder-free culture system for the efficient differentiation of multifunctional hepatocytes from human embryonic stem (ES) cells and three entirely different induced pluripotent stem (iPS) cells (including vector/transgene-free iPS cells generated using Sendai virus vector) without cell sorting and gene manipulation. The differentiation-inducing protocol consisted of a first stage; endoderm induction, second stage; hepatic initiation, and third stage; hepatic maturation. At the end of differentiation culture, hepatocytes induced from human pluripotent stem cells expressed hepatocyte-specific proteins, such as alpha-fetoprotein, albumin, alpha 1 antitrypsin and cytochrome P450 (CYP3A4), at similar or higher levels compared with three control human hepatocyte or hepatic cell lines. These human iPS/ES cell-derived hepatocytes also showed mature hepatocyte functions: inclocyanine green dye uptake (similar to 30%), storage of glycogen (>80%) and metabolic activity of CYP3A4. Furthermore, they produced a highly sensitive hepatotoxicity assay system for D-galactosamine as determined by the extracellular release of hepatocyte-specific enzymes. Hepatoprotective prostaglandin El attenuated this toxicity. Interestingly, bile duct-specific enzymes were also detected after drug treatment, suggesting the presence of bile-duct epithelial cells (cholangiocytes) in our culture system. Electron microscopic studies confirmed the existence of cholangiocytes, and an immunostaining study proved the presence of bipotential hepatoblasts with high potential for proliferation. Differentiated cells were transferrable onto new dishes, on which small-sized proliferating cells with hepatocyte markers emerged and expanded. Thus, our differentiation culture system provides mature functional hepatocytes, cholangiocytes, and their progenitors with proliferative potential from a wide variety of human pluripotent stem cells.

DOI： 10.1089/cell.2011.0064

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記述言語：英語   出版者・発行元：(NPO)日本小児がん学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BIOMED CENTRAL LTD  

Background: Human iPS cells (hiPSCs) have attracted considerable attention for applications to drug screening and analyses of disease mechanisms, and even as next generation materials for regenerative medicine. Genetic reprogramming of human somatic cells to a pluripotent state was first achieved by the ectopic expression of four factors (Sox2, Oct4, Klf4 and c-Myc), using a retrovirus. Subsequently, this method was applied to various human cells, using different combinations of defined factors. However, the transcription factor-induced acquisition of replication competence and pluripotency raises the question as to how exogenous factors induce changes in the inner and outer cellular states.
Results: We analyzed both the RNA profile, to reveal changes in gene expression, and the glycan profile, to identify changes in glycan structures, between 51 cell samples of four parental somatic cell (SC) lines from amniotic mesodermal, placental artery endothelial, and uterine endometrium sources, fetal lung fibroblast (MRC-5) cells, and nine hiPSC lines that were originally established. The analysis of this information by standard statistical techniques combined with a network approach, named network screening, detected significant expression differences between the iPSCs and the SCs. Subsequent network analysis of the gene expression and glycan signatures revealed that the glycan transfer network is associated with known epitopes for differentiation, e. g., the SSEA epitope family in the glycan biosynthesis pathway, based on the characteristic changes in the cellular surface states of the hiPSCs.
Conclusions: The present study is the first to reveal the relationships between gene expression patterns and cell surface changes in hiPSCs, and reinforces the importance of the cell surface to identify established iPSCs from SCs. In addition, given the variability of iPSCs, which is related to the characteristics of the parental SCs, a glycosyltransferase expression assay might be established to define hiPSCs more precisely and thus facilitate their standardization, which are important steps towards the eventual therapeutic applications of hiPSCs.

DOI： 10.1186/1752-0509-5-S1-S17

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

Using dodecyl N-acetylglucosaminide (GlcNAc-C12) as a saccharide primer, we investigated the biosynthetic changes of neolacto-series glycosphingolipids (GSLs) in mouse embryonal carcinoma F9 cells during differentiation induced by retinoic acid plus dibutyryl cyclic AMP (RA/dbcAMP). In the differentiated cells, the glycosylation of GlcNAc-C12 was greatly enhanced. The sugar compositions of glycosylated primers were assigned as Hex-GlcNAc, [Hex](2)-GlcNAc, [Hex](2)[HexNAc]-GlcNAc, and [NeuAc][Hex]-GlcNAc by liquid chromatography-tandem mass spectrometry. The detection of augmented biosynthesis of endogenous sialylparagloboside indicated that [NeuAc][Hex]-GlcNAc was predicted to be the non-reducing end trisaccharide of sialylparagloboside. The transcription of B3gnt5, B4galt1, Ggta1, Fut4 and St3gal6, encoding glycosyltransferases involved in the neolacto-series glycosphingolipids biosynthesis, was increased, whereas that of Fut9 and St6galI was decreased after RA/dbcAMP treatment. Furthermore, the sialyltransferase activity of ST3GalVI sialylating paragloboside was enhanced with the increase in St3gal6 expression. Since most stage-specific embryonic antigen-1 (SSEA-1) active determinants are carried by glycoproteins in F9 cells, the changes in glycolipid metabolism do not seem to be closely related to loss of cell surface SSEA-1 expression upon F9 differentiation. These results indicate that RA/dbcAMP treatment activates the biosynthesis of neolacto-series GSL and enhances sialylation of paragloboside in F9 cells with down-regulation of Fut9 expression.

DOI： 10.1093/jb/mvq142

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MARY ANN LIEBERT, INC  

Here, we report the highly efficient in vitro differentiation of human bone marrow-derived mesenchymal stem/progenitor cells (MPCs) using a novel nanotechnology-based culture plate, nanoculture plate (R) (NCP). The NCP contains uneven microfabrications with diameters of similar to 2-3 mm arranged in a honeycomb pattern on its culture surface, which is devoid of animal-derived protein sources. When human MPCs were subjected to three-dimensional (3D) culture using an NCP, they rapidly formed adhesive spheroids. We showed that adipogenic differentiation in NCP-mediated 3D cultures led to more rapid accumulation of triglycerides than that in two-dimensional cultures. During adipogenesis in 3D cultures, the rapid and intense induction of adipocyte-specific gene expressions, such as peroxisome proliferator-activated receptor gamma (PPAR-gamma), CCAAT-enhancer-binding protein alpha (C/EBP-alpha), adipocyte protein 2 (aP2), and adiponectin was observed. Together, these results indicate that this 3D culture system is suitable for the differentiation of human MPCs into adipogenic lineage, and could be applicable to adipose tissue engineering under xeno-free condition.

DOI： 10.1089/ten.tea.2009.0810

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed a-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

DOI： 10.1038/mt.2010.241

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPANDIDOS PUBL LTD  

It is hoped that the gangliosides contained in neuroblastomas (NBs) can be used as outcome predictors. We used liquid chromatography-tandem mass spectrometry (LC-MS) to analyze the gangliosides expressed in 11 NB cell lines. LC-MS analysis detected a number of gangliosides, including acetylated forms, with significantly higher sensitivity than conventional high-performance thin-layer chromatography analysis, and the results revealed that the expression profiles of the gangliosides GD1a, GD2, and acetylated GD2 differed according to the NB cell line. Hierarchical clustering based on the ganglioside expression profiles obtained by LC-MS analysis revealed that the NB cell lines could be classified into three types according to their expression of these three gangliosides: A-type characterized by high expression of GD1a and low or no expression of GD2/acetylated GD2, B-type characterized by low or no expression of GD1a and high expression of GD2/acetylated GD2, and AB-type characterized by expression of both GD1a and GD2/acetylated GD2. Interestingly, all three MYCN non-amplified cell lines were classified into the A-type. The classification was found to be correlated with mRNA expression of ganglioside synthase and neural-differentiation-related genes. The results of this study indicate that LC-MS analysis is useful as a tool for glycosphingolipid research on malignancies.

DOI： 10.3892/ijo_00000779

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.09.085

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Background: Human induced pluripotent stem (iPS) cells are currently used as powerful resources in regenerative medicine. During very early developmental stages, DNA methylation decreases to an overall low level at the blastocyst stage, from which embryonic stem cells are derived. Therefore, pluripotent stem cells, such as ES and iPS cells, are considered to have hypo-methylated status compared to differentiated cells. However, epigenetic mechanisms of "stemness'' remain unknown in iPS cells derived from extra-embryonic and embryonic cells.
Methodology/Principal Findings: We examined genome-wide DNA methylation (24,949 CpG sites covering 1,3862 genes, mostly selected from promoter regions) with six human iPS cell lines derived from human amniotic cells and fetal lung fibroblasts as well as two human ES cell lines, and eight human differentiated cell lines using Illumina's Infinium HumanMethylation27. A considerable fraction (807 sites) exhibited a distinct difference in the methylation level between the iPS/ES cells and differentiated cells, with 87.6% hyper-methylation seen in iPS/ES cells. However, a limited fraction of CpG sites with hypo-methylation was found in promoters of genes encoding transcription factors. Thus, a group of genes becomes active through a decrease of methylation in their promoters. Twenty-three genes including SOX15, SALL4, TDGF1, PPP1R16B and SOX10 as well as POU5F1 were defined as genes with hypo-methylated SS-DMR (Stem cell-Specific Differentially Methylated Region) and highly expression in iPS/ES cells.
Conclusions/Significance: We show that DNA methylation profile of human amniotic iPS cells as well as fibroblast iPS cells, and defined the SS-DMRs. Knowledge of epigenetic information across iPS cells derived from different cell types can be used as a signature for "stemness'' and may allow us to screen for optimum iPS/ES cells and to validate and monitor iPS/ES cell derivatives for human therapeutic applications.

DOI： 10.1371/journal.pone.0013017

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC HEMATOLOGY  

LMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid-rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA-induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL. (Blood. 2010;116(6):962-970)

DOI： 10.1182/blood-2009-09-244673

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Baculovirus expression systems (BES) are widely used for recombinant protein production in lepidopteran cells or larvae. However, even in BES, the insolubility of recombinant proteins sometimes makes their expression difficult. In this study, to improve the solubility and yield of foreign proteins, we constructed transgenic silkworms using silkworm heat-shock proteins, Hsp70 and Hsp40, or Hsc70 and Hsp90 co-chaperone Hop. In these transgenic silkworms, the expression levels of the transgenes were under the control of a UAS center dot hsp mini-promoter driven by a Gal4NFkBp65 activator. When the transgenic silkworm with HSP70 and 40 (TGS-HSP70/40) was infected with BmNPV carrying mC3d and Gal4NFkBp65 under the control of baculovirus polyhedrin or p10 promoters, respectively, the soluble fraction of the His- or His center dot GST-tagged mC3d increased significantly. Similarly, the transgenic silkworm with HSC70 and HOP (TGS-HOP7) was effective for the expression of a steroid hormone receptor, USP2. In conclusion, the His-tagged baculovirus expression system featuring the chaperone effect TGS-HSP70/40 and TGS-HOP7 silkworms is effective for increasing the yields of soluble and functional foreign gene products.

DOI： 10.1007/s00253-010-2617-0

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：SPRINGER  

DOI： 10.1007/978-90-481-3892-0_16

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

Practical clinical applications for current induced pluripotent stem cell (iPSC) technologies are hindered by very low generation efficiencies. Here, we demonstrate that newborn human (h) and mouse (m) extra-embryonic amnion (AM) and yolk-sac (YS) cells, in which endogenous KLF4/Klf4, c-MYC/c-Myc and RONIN/Ronin are expressed, can be reprogrammed to hiPSCs and miPSCs with efficiencies for AM cells of 0.02% and 0.1%, respectively. Both hiPSC and miPSCs are indistinguishable from embryonic stem cells in colony morphology, expression of pluripotency markers, global gene expression profile, DNA methylation status of OCT4 and NANOG, teratoma formation and, in the case of miPSCs, generation of germline transmissible chimeric mice. As copious amounts of human AM cells can be collected without invasion, and stored long term by conventional means without requirement for in vitro culture, they represent an ideal source for cell banking and subsequent 'on demand' generation of hiPSCs for personal regenerative and pharmaceutical applications.

DOI： 10.1111/j.1365-2443.2009.01356.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1365-2567.2009.03122.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

P>With an increase in the importance of umbilical cord blood (CB) as an alternative source of haematopoietic progenitors for allogenic transplantation, donor lymphocyte infusion (DLI) with donor CB-derived activated CD4+ T cells in the unrelated CB transplantation setting is expected to be of increased usefulness as a direct approach for improving post-transplant immune function. To clarify the characteristics of activated CD4+ T cells derived from CB, we investigated their mRNA expression profiles and compared them with those of peripheral blood (PB)-derived activated CD4+ T cells. Based on the results of a DNA microarray analysis and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), a relatively high level of forkhead box protein 3 (Foxp3) gene expression and a relatively low level of interleukin (IL)-17 gene expression were revealed to be significant features of the gene expression profile of CB-derived activated CD4+ T cells. Flow cytometric analysis further revealed protein expression of Foxp3 in a portion of CB-derived activated CD4+ T cells. The low level of retinoic acid receptor-related orphan receptor gamma isoform t (ROR gamma t) gene expression in CB-derived activated CD4+ T cells was speculated to be responsible for the low level of IL-17 gene expression. Our data indicate a difference in gene expression between CD4+ T cells from CB and those from PB. The findings of Foxp3 expression, a characteristic of regulatory T cells, and a low level of IL-17 gene expression suggest that CB-derived CD4+ T cells may be a more appropriate source for DLI.

DOI： 10.1111/j.1365-2567.2009.03122.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER INC  

POU5F1 (more commonly known as OCT4/3) is one of the stern cell markers, and affects direction of differentiation in embryonic stern cells. To investigate whether cells of mesenchymal origin acquire embryonic phenotypes, we generated human cells of mesodermal origin with overexpression of the chimeric OCT4/3 gene with physiological co-activator EWS (product of the EWSR1 gene), which is driven by the potent EWS promoter by translocation. The cells expressed embryonic stern cell genes such as NANOG, lost mesenchymal phenotypes, and exhibited embryonal stern cell-like alveolar structures when implanted into the subcutaneous tissue of immunodeficient mice. Hierarchical analysis by microchip analysis and Cell surface analysis revealed that the cells are subcatagorized into the group Of embryonic stern cells and embryonal Carcinoma cells. These results imply that cells of mesenchymal origin call be traced back to cells of embryonic phenotype by the OCT4/3 gene in collaboration with the potent cis-regulatory element and the fused co-activator. The cells generated in this study with overexpression of chimeric OCT4/3 provide Lis with insight into cell plasticity involving OCT4/3 that is essential for embryonic cell maintenance, and the complexity required for changing cellular identity. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2009.06.016

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MARY ANN LIEBERT INC  

The CH-296 recombinant fragment of human fibronectin is essential for murine leukemia virus (MLV)-derived retroviral transduction of CD34(+) cells for the purpose of stem cell gene therapy. Although the major effect of CH-296 is colocalization of the MLV-derived retrovirus and target cells at specific adhesion domains of CH-296 mediated by integrins expressed on CD34(+) cells, the precise roles of the integrins are unclear. We examined the kinetics of integrin expression on CD34(+) cells during the course of MLV-derived retrovirus-mediated gene transduction with CH-296. Flow cytometry revealed that the levels of both very late activation protein (VLA)-4 and VLA-5 on CD34(+) cells freshly isolated from cord blood were insufficient for effective MLV-derived retroviral transduction. However, increases were achieved during culture for preinduction and MLV-derived retrovirus-mediated gene transduction in the presence of a cocktail of cytokines. In addition, we confirmed by using specific antibodies that inhibition of the cell adhesion mediated by the integrins significantly reduced transduction efficiency, indicating that integrin expression is indeed important for CH-296-based MLV-derived retroviral transduction. Only a few cytokines are capable of inducing integrin expression, and stem cell factor plus thrombopoietin was found to be the minimal combination that was sufficient for effective transduction of an MLV-derived retrovirus based on CH-296. Our findings should be useful for improving the culture conditions for CH-296-based MLV-derived retroviral transduction in stem cell gene therapy.

DOI： 10.1089/hum.2008.159

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

The expression of granulysin, a cytolytic protein produced by activated T and NK cells, has been revealed to be correlated with the prognosis of some adult cancer patients. By examination on various childhood lymphoma tissues, we found that granulysin level was especially high in systemic anaplastic large cell lymphoma (ALCL) cases, whereas no close correlation with the expression of CD96, a marker for activated T and NK cells, was observed. We further demonstrated that both ALCL cells in biopsy specimens and cell lines established from ALCL express granulysin, indicating some correlation of gramulysin with biological features of ALCL (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.leukres.2009.01.032

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Background: The Dickkopf (DKK) family comprises a set of proteins that function as regulators of Wnt/beta-catenin signaling and has a crucial role in development. Recent studies have revealed the involvement of this family in tumorigenesis, however their role in tumorigenesis is still remained unclear.
Methodology/Principal Findings: We found increased expression of DKK2 but decreased expression of DKK1 in Ewing family tumor (EFT) cells. We showed that EFT-specific EWS/ETS fusion proteins enhance the DKK2 promoter activity, but not DKK1 promoter activity, via ets binding sites (EBSs) in the 5' upstream region. EWS/ETS-mediated transactivation of the promoter was suppressed by the deletion and mutation of EBSs located upstream of the DKK2 gene. Interestingly, the inducible expression of EWS/ETS resulted in the strong induction of DKK2 expression and inhibition of DKK1 expression in human primary mesenchymal progenitor cells that are thought to be a candidate of cell origin of EFT. In addition, using an EFT cell line SK-ES1 cells, we also demonstrated that the expression of DKK1 and DKK2 is mutually exclusive, and the ectopic expression of DKK1, but not DKK2, resulted in the suppression of tumor growth in immuno-deficient mice.
Conclusions/Significance: Our results suggested that DKK2 could not functionally substitute for DKK1 tumor-suppressive effect in EFT. Given the mutually exclusive expression of DKK1 and DKK2, EWS/ETS regulates the transcription of the DKK family, and the EWS/ETS-mediated DKK2 up-regulation could affect the tumorigenicity of EFT in an indirect manner.

DOI： 10.1371/journal.pone.0004634

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

B-cell-activating factor (BAFF) is a survival and maturation factor for B cells belonging to the tumour necrosis factor superfamily. Among three identified functional receptors, the BAFF receptor (BAFF-R) is thought to be responsible for the effect of BAFF on B cells though details of how remain unclear. We determined that a hairy-cell leukaemia line, MLMA, expressed a relatively high level of BAFF-R and was susceptible to apoptosis mediated by either CD20 or B-cell antigen receptor (BCR). Using MLMA cells as an in vitro model of mature B cells, we found that treatment with BAFF could inhibit apoptosis mediated by both CD20 and BCR. We also observed, using immunoblot analysis and microarray analysis, that BAFF treatment induced activation of nuclear factor-kappa B2 following elevation of the expression level of Bcl-2, which may be involved in the molecular mechanism of BAFF-mediated inhibition of apoptosis. Interestingly, BAFF treatment was also found to induce the expression of a series of genes, such as that for CD40, related to cell survival, suggesting the involvement of a multiple mechanism in the BAFF-mediated anti-apoptotic effect. MLMA cells should provide a model for investigating the molecular basis of the effect of BAFF on B cells in vitro and will help to elucidate how B cells survive in the immune system in which BAFF-mediated signalling is involved.

DOI： 10.1111/j.1365-2567.2008.02872.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Detergent-insoluble microdomains, or rafts, act as a platform to transduce signals from the extracellular space into the cytoplasm. In the process of developing monoclonal antibodies against raft molecules for the purpose of studying the molecular mechanism of raft-mediated signaling, we observed the uniqueness and certain advantages of immunization with rafts. Simple subcutaneous injection of mice with a phosphate-buffered saline (PBS) suspension of rafts without mixing with Freund's adjuvant made it possible to increase the titer of antiserum reacting with raft components. Interestingly, injection of rafts prepared from certain specific cell lines induced monoglycolipid-specific antibodies. Furthermore, antibodies were produced by raft-immunization of even syngeneic mice. Our findings suggest that this phenomenon does not represent a breakdown of immunological self-tolerance, but typical immune reactions accompanying the class switch from IgM antibodies to IgG antibodies.

DOI： 10.1007/s10719-007-9083-7

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記述言語：英語  

DOI： 10.1128/MCB.00557-08

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

Ewing's family tumor (EFT) is a rare pediatric tumor of unclear origin that occurs in bone and soft tissue. Specific chromosomal translocations found in EFT cause EWS to fuse to a subset of ets transcription factor genes (ETS), generating chimeric EWS/ETS proteins. These proteins are believed to play a crucial role in the onset and progression of EFT. However, the mechanisms responsible for the EWS/ETS-mediated onset remain unclear. Here we report the establishment of a tetracycline-controlled EWS/ETS-inducible system in human bone marrow-derived mesenchymal progenitor cells (MPCs). Ectopic expression of both EWS/FLI1 and EWS/ERG proteins resulted in a dramatic change of morphology, i.e., from a mesenchymal spindle shape to a small round-to-polygonal cell, one of the characteristics of EFT. EWS/ETS also induced immunophenotypic changes in MPCs, including the disappearance of the mesenchyme-positive markers CD10 and CD13 and the up-regulation of the EFT-positive markers CD54, CD99, CD117, and CD271. Furthermore, a prominent shift from the gene expression profile of MPCs to that of EFT was observed in the presence of EWS/ETS. Together with the observation that EWS/ETS enhances the ability of cells to invade Matrigel, these results suggest that EWS/ETS proteins contribute to alterations of cellular features and confer an EFT-like phenotype to human MPCs.

DOI： 10.1128/MCB.00740-07

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The monoclonal antibody 6E2 raised against the embryonal carcinoma cell line NCR-G3 had been shown to also react with human germ cells. Thin-layer chromatography (TLC) immunostaining revealed that 6E2 specifically reacts with sialosylglobopentaosylceramide (sialylGb5), which carries an epitope of stage-specific embryonic antigen-4 (SSEA-4), known as an important cell surface marker of embryogenesis. The immunostaining of mouse preimplantation embryos without fixation showed that the binding of 6E2 caused the clustering and consequent accumulation of sialylGb5 at the interface between blastomeres. These results suggest that SSEA-4 actively moves on the cell surface and readily accumulates between blastomeres after binding of 6E2. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.10.093

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記述言語：英語   出版者・発行元：Japanese Society of Sericultural Science  

2007, Oct

DOI： 10.11416/jibs.76.3_137

CiNii Books

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE INC  

Objective. The role of interleukin (IL)-7 in human B lymphopoiesis is still controversial. We used an in vitro culture system to verify involvement of IL-7 in development of human pro-B cells from hematopoietic stem cells.
Materials and Methods. Human CD34(+) bone marrow cells were cultured for 4 weeks on MS-5 mouse strornal cells to induce pro-B cells. Expression of IL-7 receptor alpha or other B-cell differentiation marker genes on cultured human CD34(+)bone marrow cells was investigated by reverse transcription polymerase chain reaction (RT-PCR). Colony assay of human CD34(+) bone marrow cells was also performed to determine the effect of IL-7 on colony-forming ability. Neutralizing antibody or reagent that eliminates the effect of IL-7 was added to the culture system, and the number of pro-B cells induced was estimated by flow cytometry.
Results. RT-PCR analysis revealed mRNA expression of IL-7 receptor a as well as B-cell differentiation marker genes in not only CD19(+) pro-B cells but also CD19(-) CD33(-) cells induced from CD34(+) bone marrow cells after cultivation for 4 weeks on MS-5 cells. Addition of anti-mouse IL-7 antibody, anti-human IL-7 receptor alpha antibody, or JAK3 kinase inhibitor reduced the number of pro-B cells induced, demonstrating that elimination of IL-7 reduces pro-B-cell development. Addition of anti-mouse IL-7 antibody emphasized the colony-forming ability of burst-forming unit erythroid cells.
Conclusions. IL-7 produced by MS-5 cells is required for human pro-B-cell development from CD34(+)bone marrow cells in our culture system, and IL-7 appears to play a certain role in early human B lymphopoiesis. (c) 2007 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

DOI： 10.1016/j.exphem.2007.05.019

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The AHA1 (activator of Hsp90 ATPase) family of proteins were exclusively conserved from yeast to humans, but little is known about their tissue distribution or biological function. In this study, a cDNA for a Bombyx mori AHA1 homologue, BmAHA1, was isolated from the testes of larvae on day 3 of the fifth instar using an mRNA differential display method.This cDNA encodes a protein with 341 amino acid residues. Gene expression studies revealed that BmAHA1 mRNA occurred prominently in the testes. In situ hybridization and immunostaining showed that the BmAHA1 mRNA signals were strongly detected in spermatogonial cells and primary spermatocytes at the fifth larval instar stage, whereas the BmAha1 protein was abundant in round and elongated spermatids at the pupal stage. The localization pattern of the accumulated protein in the elongated spermatids was reminiscent of that reported previously for microtubules, but the BmAha1 protein showed a decrease in apparent concentration during maturation process. The stage-and cell-specific expression indicated that BmAha1 might play a role in silkworm spermatogenesis, especially in postmeiotic differentiation. © 2005 The Royal Entomological Society.

DOI： 10.1111/j.1365-2583.2005.00553.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Nonhomologous end-Joining (NHEJ) is one of the repair pathways for double-strand breaks (DSBs) in eukaryotic cells. By using linearized plasmid substrates, we have detected intramolecular NHEJ activity in a cell-free extract from the cultured silkworm cell line BmN4. The efficiency of NHEJ differed according to the structure of DNA ends; approximately 1% of input DNA was repaired when the substrate had cohesive ends. The reaction required the hydrolysis of nucleotide triphosphate; interestingly, all of four rNTPs or four dNTPs could support the reaction. A substrate with non-complementary DNA ends was mainly repaired by the DNA polymerase-mediated pathway. These results indicate that the present cell-free system will be useful to analyze the molecular mechanisms of DSB repair and NHEJ in insect cells.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

The ribonuclease L ( RNase L) pathway plays an important role in the response of cells to double-stranded RNA ( dsRNA) during the events such as virus infection. Ribonuclease L inhibitor (RLI) belonging to the ABC transporter family is known as a regulator of the RNase L pathway. The homologs of RLI were reported in many organisms including the fruit fly and mosquito, but their functions in insects and arthropods have not been elucidated to date. In the present study, we cloned a cDNA of a silkworm RLI homolog, termed BmRLI, and its nucleotide sequence was determined. RT-PCR analysis revealed that the expression of BmRLI mRNA was marked in the testis, ovary and fat body. From the cDNA, recombinant protein with an apparent molecular mass of 69 kDa was expressed in Escherichia coli and cultured insect cells. Although no obvious effect of up-regulation of the BmRLI expression on RNAi was observed, its down-regulation slightly reduced RNAi efficiency.

DOI： 10.1080/10425170400028871

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DOI： 10.11416/jibs.74.21

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DOI： 10.1016/j.cbpc.2004.06.004

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DOI： 10.11416/jibs.73.117

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記述言語：日本語  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CELL PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：NATURE PUBLISHING GROUP  

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記述言語：英語   出版者・発行元：NATURE PUBLISHING GROUP  

DOI： 10.1038/mt.2014.216

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：NATURE PUBLISHING GROUP  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JOHN WILEY & SONS LTD  

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開催地：Osaka, Japan  

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開催地：Fukuoka, Japan  

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開催地：Yokohama, Japan  

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開催地：Yokohama, Japan  

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開催地：Sendai, Japan  

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開催地：Sendai, Japan  

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開催地：Sendai, Japan  

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開催地：Sendai, Japan  

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開催地：Yokohama, Japan  

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開催地：Yokohama, Japan  

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開催地：Yokohama, Japan  

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開催地：Tokyo, Japan  

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開催地：Tokyo, Japan  

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開催地：Kanazawa, Japan  

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開催地：San Diego, USA  

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開催地：Chiba, Japan  

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開催地：Kumamoto, Japan  

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開催地：Sangju, Korea  

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開催地：Kyoto, Japan  

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開催地：Tokyo, Japan  

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開催地：Chiba, Japan  

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開催地：Nagoya, Japan  

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開催地：Chiba, Japan  

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開催地：Kobe, Japan  

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開催地：Kobe, Japan  

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開催地：Kobe, Japan  

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開催地：Kobe, Japan  

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開催地：Fukuoka, Japan  

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開催地：Osaka, Japan  

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開催地：Suzhou, China  

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開催地：Tokyo, Japan  

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開催地：Maehashi, Japan  

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開催地：Tokyo, Japan  

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開催地：Maehashi, Japan  

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開催地：Yokohama, Japan,  

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開催地：Yokohama, Japan  

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開催地：Kobe Japan  

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開催地：Yokohama, Japan  

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開催地：Kobe, Japan  

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開催地：Kobe Japan  

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開催地：Washington, DC, USA  

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開催地：Oxford, UK  

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開催地：Kobe, Japan  

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開催地：New Orleans, USA  

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開催地：Washington, DC、USA  

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開催地：Kyoto, Japan  

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開催地：Yokohama. Japan  

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開催地：Tokyo, Japan  

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開催地：Okayama, Japan  

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開催地：Yokohama. Japan  

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開催地：Tokyo Japan  

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開催地：Okayama, Japan  

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開催地：Kobe, Japan  

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開催地：Kobe, Japan  

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開催地：Chicago, USA  

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開催地：Kobe, Japan  

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開催地：Yokohama. Japan  

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開催地：Tokyo, Japan  

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開催地：Kobe, Japan  

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開催地：Okayama, Japan  

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開催地：Fukuoka, Japan  

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開催地：Yokohama, Japan  

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開催地：Kyoto, Japan  

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開催地：Kyoto, Japan  

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開催地：Kobe, Japan  

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開催地：Kobe, Japan  

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開催地：Kyoto, Japan  

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開催地：Kyoto, Japan  

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開催地：Yokohama, Japan  

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開催地：Kyoto, Japan  

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開催地：Osaka, Japan  

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