論文 - 岡野　栄之

Low immunogenicity of mouse induced pluripotent stem cell-derived neural stem cells

Itakura G, Ozaki M, Nagoshi N, Kawabata S, Nishiyama Y, Sugai K, Iida T, Rei Kashiwagi R, Ookubo T, Yastake K, Matsubayashi K, Kohyama J, Iwanami A, Matsumoto M, Nakamura M, and Okano H

Sci Rep 7   12996 2017年

RNA-binding protein Qki5 regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling

Hayakawa-Yano Y, Suyama S, Nogami M, Yugami M, Koya I, Furukawa T, Zhou L,Abe M, Sakimura K, Takebayashi H, Nakanishi A, Okano H*, and Yano M.*

Genes & Dev 2017年

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Assessment of mitophagy in iPS cell-derived neurons

Ishikawa K., Yamaguchi A., Okano H., Akamatsu W.

Methods in Molecular Biology （Methods in Molecular Biology）  1759   59 - 67 2017年

ISSN  10643745

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© 2017, Springer Science+Business Media New York. Aberrant mitochondrial function is associated with many neurological diseases. Mitophagy is a key mechanism for the elimination of damaged mitochondria and maintenance of mitochondrial homeostasis. Induced pluripotent stem (iPS) cell technologies developed over the last decade have allowed us to analyze functions of the human neuron. Here we describe an efficient induction method from human iPS cells to neurons, followed by an image-based mitophagy assay.

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STAT3: Down the R(h)oAd

Renault-Mihara F., Okano H.

Cytokine （Cytokine）  102   149 - 150 2017年

ISSN  10434666

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STAT3-regulated RhoA drives reactive astrocyte dynamics

Renault-Mihara F, Okano H

Cell Cycle 2017年

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iPSC-derived neural precursor cells: potential for cell transplantation therapy in spinal cord injury

Nagoshi N., Okano H.

Cellular and Molecular Life Sciences （Cellular and Molecular Life Sciences）  75 （ 6 ） 989 - 1000 2017年

ISSN  1420682X

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© 2017, Springer International Publishing AG. A number of studies have demonstrated that transplantation of neural precursor cells (NPCs) promotes functional recovery after spinal cord injury (SCI). However, the NPCs had been mostly harvested from embryonic stem cells or fetal tissue, raising the ethical concern. Yamanaka and his colleagues established induced pluripotent stem cells (iPSCs) which could be generated from somatic cells, and this innovative development has made rapid progression in the field of SCI regeneration. We and other groups succeeded in producing NPCs from iPSCs, and demonstrated beneficial effects after transplantation for animal models of SCI. In particular, efficacy of human iPSC–NPCs in non-human primate SCI models fostered momentum of clinical application for SCI patients. At the same time, however, artificial induction methods in iPSC technology created alternative issues including genetic and epigenetic abnormalities, and tumorigenicity after transplantation. To overcome these problems, it is critically important to select origins of somatic cells, use integration-free system during transfection of reprogramming factors, and thoroughly investigate the characteristics of iPSC–NPCs with respect to quality management. Moreover, since most of the previous studies have focused on subacute phase of SCI, establishment of effective NPC transplantation should be evaluated for chronic phase hereafter. Our group is currently preparing clinical-grade human iPSC–NPCs, and will move forward toward clinical study for subacute SCI patients soon in the near future.

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Mapping orbitofrontal-limbic maturation in non-human primates: A longitudinal magnetic resonance imaging study

Uematsu A, Hata J, Komaki Y, Seki F, Yamada C, Okahara N, Kurotaki Y, Sasaki E, Okano H

Neuroimage 163   55 - 67 2017年

RNA Sequencing Analysis Reveals Interactions between Breast Cancer or Melanoma Cells and the Tissue Microenvironment during Brain Metastasis

Sato Ryo, Nakano Teppei, Hosonaga Mari, Sampetrean Oltea, Harigai Ritsuko, Sasaki Takashi, Koya Ikuko, Okano Hideyuki, Kudoh Jun, Saya Hideyuki, Arima Yoshimi

BioMed Research International 2017 2017年

ISSN  2314-6133

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<p>Metastasis is the main cause of treatment failure and death in cancer patients. Metastasis of tumor cells to the brain occurs frequently in individuals with breast cancer, non-small cell lung cancer, or melanoma. Despite recent advances in our understanding of the causes and in the treatment of primary tumors, the biological and molecular mechanisms underlying the metastasis of cancer cells to the brain have remained unclear. Metastasizing cancer cells interact with their microenvironment in the brain to establish metastases. We have now developed mouse models of brain metastasis based on intracardiac injection of human breast cancer or melanoma cell lines, and we have performed RNA sequencing analysis to identify genes in mouse brain tissue and the human cancer cells whose expression is associated specifically with metastasis. We found that the expressions of the mouse genes Tph2, Sspo, Ptprq, and Pole as well as those of the human genes CXCR4, PLLP, TNFSF4, VCAM1, SLC8A2, and SLC7A11 were upregulated in brain tissue harboring metastases. Further characterization of such genes that contribute to the establishment of brain metastases may provide a basis for the development of new therapeutic strategies and consequent improvement in the prognosis of cancer patients.</p>

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Plasticity and regeneration in the injured spinal cord after cell transplantation therapy

Nori S., Nakamura M., Okano H.

Progress in Brain Research 2017年

ISSN  0079-6123

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<p>Spinal cord injury (SCI) typically damages the long axonal tracts of the spinal cord which results in permanent disability. However, regeneration of the injured spinal cord is approaching reality according to the advances in stem cell biology. Cell transplantation therapy holds potential to lead to recovery following SCI through some positive mechanisms. Grafted cells induce plasticity and regeneration in the injured spinal cord by promoting remyelination of damaged axons, reconstruction of neural circuits by synapse formation between host neurons and graft-derived neurons, and secreting neurotrophic factors to promote axonal elongation as well as reduce retrograde axonal degeneration. In this review, we will delineate (1) the microenvironment of the injured spinal cord that influence the plasticity and regeneration capacity after SCI, (2) a number of different kinds of cell transplantation therapies for SCI that has been extensively studied by researchers, and (3) potential mechanisms of grafted cell-induced regeneration and plasticity in the injured spinal cord.</p>

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Brief exposure to small molecules allows induction of mouse embryonic fibroblasts into neural crest-like precursors

Takayama Yuzo, Wakabayashi Tamami, Kushige Hiroko, Saito Yutaka, Shibuya Yoichiro, Shibata Shinsuke, Akamatsu Wado, Okano Hideyuki, Kida Yasuyuki S.

FEBS Letters 2017年

ISSN  0014-5793

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<p>In this study, we propose a novel method for inducing neuronal cells by briefly exposing them to small-molecule cocktails in a step-by-step manner. Global gene expression analysis with immunohistochemical staining and calcium flux assays reveal the generation of neurons from mouse embryonic fibroblasts. In addition, time-lapse imaging of neural precursor-specific enhancer expression and global gene expression analyses show that the neurons are generated by passing through a neural crest-like precursor stage. Consistent with these results, the neural crest-like cells are able to differentiate into neural crest lineage cells, such as sympathetic neurons, adipocytes, osteocytes, and smooth muscle cells. Therefore, these results indicate that brief exposure to chemical compounds could expand and induce a substantial multipotent cell population without viral transduction.</p>

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Differential X chromosome inactivation patterns during the propagation of human induced pluripotent stem cells

Andoh-Noda Tomoko, Akamatsu Wado, Miyake Kunio, Kobayashi Tetsuro, Ohyama Manabu, Kurosawa Hiroshi, Kubota Takeo, Okano Hideyuki

Keio Journal of Medicine 66 （ 1 ） 1 - 8 2017年

ISSN  0022-9717

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<p>Human induced pluripotent stem cells (hiPSCs) represent a potentially useful tool for studying te molecular mechanisms of disease thanks to their ability to generate patient-specific hiPSC clones. However, previous studies have reported that DNA methylation profiles, including those for imprinted genes, may change during passaging of hiPSCs. This is particularly problematic for hiPSC models of X-linked disease, because unstable X chromosome inactivation status may affect the detection of phenotypes. In the present study, we examined the epigenetic status of hiPSCs derived from patients with Rett syndrome, an X-linked disease, during long-term culture. To analyze X chromosome inactivation, we used a methylation-specific polymerase chain reaction (MSP) to assay the human androgen receptor locus (HUMARA). We found that single cell-derived hiPSC clones exhibit various states of X chromosome inactivation immediately after clonal isolation, even when established simultaneously from a single donor. X chromosome inactivation states remain variable in hiPSC clones at early passages, and this variability may affect cellular phenotypes characteristic of X-linked diseases. Careful evaluation of X chromosome inactivation in hiPSC clones, particularly in early passages, by methods such as HUMARA-MSP, is therefore important when using patient-specific hiPSCs to model X-linked disease.</p>

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Whole-Genome DNA Methylation Analyses Revealed Epigenetic Instability in Tumorigenic Human iPS Cell-Derived Neural Stem/Progenitor Cells

Iida Tsuyoshi, Iwanami Akio, Sanosaka Tsukasa, Kohyama Jun, Miyoshi Hiroyuki, Nagoshi Narihito, Kashiwagi Rei, Toyama Yoshiaki, Matsumoto Morio, Nakamura Masaya, Okano Hideyuki

Stem Cells 2017年

ISSN  1066-5099

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<p>Although human induced pluripotent stem cell (hiPSC) derivatives are considered promising cellular resources for regenerative medicine, their tumorigenicity potentially limits their clinical application in hiPSC technologies. We previously demonstrated that oncogenic hiPSC-derived neural stem/progenitor cells (hiPSC-NS/PCs) produced tumor-like tissues that were distinct from teratomas. To gain insight into the mechanisms underlying the regulation of tumorigenicity in hiPSC-NS/PCs, we performed an integrated analysis using the Infinium HumanMethylation450 BeadChip array and the HumanHT-12 v4.0 Expression BeadChip array to compare the comprehensive DNA methylation and gene expression profiles of tumorigenic hiPSC-NS/PCs (253G1-NS/PCs) and non-tumorigenic cells (201B7-NS/PCs). Although the DNA methylation profiles of 253G1-hiPSCs and 201B7-hiPSCs were similar regardless of passage number, the methylation status of the global DNA methylation profiles of 253G1-NS/PCs and 201B7-NS/PCs differed; the genomic regions surrounding the transcriptional start site of the CAT and PSMD5 genes were hypermethylated in 253G1-NS/PCs but not in 201B7-NS/PCs. Interestingly, the aberrant DNA methylation profile was more pronounced in 253G1-NS/PCs that had been passaged more than 15 times. In addition, we identified aberrations in DNA methylation at the RBP1 gene locus; the DNA methylation frequency in RBP1 changed as 253G1-NS/PCs were sequentially passaged. These results indicate that different NS/PC clones have different DNA methylomes and that DNA methylation patterns are unstable as cells are passaged. Therefore, DNA methylation profiles should be included in the criteria used to evaluate the tumorigenicity of hiPSC-NS/PCs in the clinical setting.</p>

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Changeability of the fully methylated status of the 15q11.2 region in induced pluripotent stem cells derived from a patient with Prader-Willi syndrome

Okuno Hironobu, Nakabayashi Kazuhiko, Abe Kousei, Ando Takayuki, Sanosaka Tsukasa, Kohyama Jun, Akamatsu Wado, Ohyama Manabu, Takahashi Takao, Kosaki Kenjiro, Okano Hideyuki

Congenital Anomalies 2017年

ISSN  0914-3505

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<p>Prader-Will syndrome (PWS) is characterized by hyperphagia, growth hormone deficiency and central hypogonadism caused by the dysfunction of the hypothalamus. Patients with PWS present with methylation abnormalities of the PWS-imprinting control region in chromosome 15q11.2, subject to parent-of-origin-specific methylation and controlling the parent-of-origin-specific expression of other paternally expressed genes flanking the region. In theory, the reversal of hypermethylation in the hypothalamic cells could be a promising strategy for the treatment of PWS patients, since cardinal symptoms of PWS patients are correlated with dysfunction of the hypothalamus. The genome-wide methylation status dramatically changes during the reprograming of somatic cells into induced pluripotent stem cells (iPSCs) and during the in vitro culture of iPSCs. Here, we tested the methylation status of the chromosome 15q11.2 region in iPSCs from a PWS patient using pyrosequencing and a more detailed method of genome-wide DNA methylation profiling to reveal whether iPSCs with a partially unmethylated status for the chromosome 15q11.2 region exhibit global methylation aberrations. As a result, we were able to show that a fully methylated status for chromosome 15q11.2 in a PWS patient could be reversed to a partially unmethylated status in at least some of the PWS-iPSC lines. Genome-wide DNA methylation profiling revealed that the partial unmethylation occurred at differentially methylated regions located in chromosome 15q11.2, but not at other differentially methylated regions associated with genome imprinting. The present data potentially opens a door to cell-based therapy for PWS patients and, possibly, patients with other disorders associated with genomic imprinting.</p>

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Applications of induced pluripotent stem cell technologies in spinal cord injury

Nagoshi Narihito, Okano Hideyuki

Journal of Neurochemistry 2017年

ISSN  0022-3042

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<p>Numerous basic research studies have suggested the potential efficacy of neural precursor cell (NPC) transplantation in spinal cord injury (SCI). However, in most such studies, the origin of the cells used was mainly fetal tissue or embryonic stem cells, both of which carry potential ethical concerns with respect to clinical use. The development of induced pluripotent stem cells (iPSCs) opened a new path toward regenerative medicine for SCI. iPSCs can be generated from somatic cells by induction of transcription factors, and induced to differentiate into NPCs with characteristics of cells of the central nervous system. The beneficial effect of iPSC-derived NPC transplantation has been reported from our group and others working in rodent and non-human primate models. These promising results facilitate the application of iPSCs for clinical applications in SCI patients. However, iPSCs also have issues, such as genetic/epigenetic abnormalities and tumorigenesis because of the artificial induction method, that must be addressed prior to clinical use. The selection of somatic cells, generation of integration-free iPSCs, and characterization of differentiated NPCs with thorough quality management are all needed to address these potential risks. To enhance the efficacy of the transplanted iPSC-NPCs, especially at chronic phase of SCI, administration of a chondroitinase or semaphorin3A inhibitor represents a potentially important means of promoting axonal regeneration through the lesion site. The combined use of rehabilitation with such cell therapy approaches is also important, as repetitive training enhances neurite outgrowth of transplanted cells and strengthens neural circuits at central pattern generators. Our group has already evaluated clinical grade iPSC-derived NPCs, and we look forward to initiating clinical testing as the next step toward determining whether this approach is safe and effective for clinical use. This article is part of the series "60th Anniversary of the Japanese Society for Neurochemistry".</p>

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Regulation of RhoA by STAT3 coordinates glial scar formation

Renault-Mihara Francois, Mukaino Masahiko, Shinozaki Munehisa, Kumamaru Hiromi, Kawase Satoshi, Baudoux Matthieu, Ishibashi Toshiki, Kawabata Soya, Nishiyama Yuichiro, Sugai Keiko, Yasutake Kaori, Okada Seiji, Nakamura Masaya, Okano Hideyuki

Journal of Cell Biology 216 （ 8 ） 2533 - 2550 2017年

ISSN  0021-9525

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<p>Understanding how the transcription factor signal transducer and activator of transcription-3 (STAT3) controls glial scar formation may have important clinical implications. We show that astrocytic STAT3 is associated with greater amounts of secreted MMP2, a crucial protease in scar formation. Moreover, we report that STAT3 inhibits the small GTPase RhoA and thereby controls actomyosin tonus, adhesion turnover, and migration of reactive astrocytes, as well as corralling of leukocytes in vitro. The inhibition of RhoA by STAT3 involves ezrin, the phosphorylation of which is reduced in STAT3-CKO astrocytes. Reduction of phosphatase and tensin homologue (PTEN) levels in STAT3-CKO rescues reactive astrocytes dynamics in vitro. By specific targeting of lesion-proximal, reactive astrocytes in Nestin-Cre mice, we show that reduction of PTEN rescues glial scar formation in Nestin-Stat3^+/- mice. These findings reveal novel intracellular signaling mechanisms underlying the contribution of reactive astrocyte dynamics to glial scar formation.</p>

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Three-dimensional kinematic and kinetic analysis of quadrupedal walking in the common marmoset (Callithrix jacchus)

Shimada Hikaru, Kanai Ryogo, Kondo Takahiro, Yoshino-Saito Kimika, Uchida Akito, Nakamura Masaya, Ushiba Junichi, Okano Hideyuki, Ogihara Naomichi

Neuroscience Research 2017年

ISSN  0168-0102

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<p>The common marmoset has recently gained a great deal of attention as an experimental primate model for biological science and medical research. To use the common marmoset for development of novel treatments and rehabilitation for locomotor disorders, it is crucial to understand fundamental baseline characteristics of locomotion in this species. Therefore, in the present study we performed kinematic and kinetic analyses of quadrupedal locomotion in this animal. A total of 14 common marmosets walking quadrupedally along a walkway were analyzed using synchronized high-speed cameras, with two force platforms set in the walkway. Our results demonstrated that the marmoset uses a lateral sequence walking pattern, in contrast to the macaque and other primates, which usually adopt a diagonal sequence pattern. Furthermore, peak vertical ground reaction force on the forelimb was larger than that on the hindlimb. The rate of energy recovery for quadrupedal walking in the common marmoset was much smaller than that in the macaque, indicating that the marmoset generally utilizes bouncing mechanics in locomotion, even though the duty factor is &gt;. 0.5. This description of locomotor characteristics of intact marmosets may serve as a basis for comparative analyses of changes in gait due to rehabilitation and regenerative treatments.</p>

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Neuroscience research using non-human primate models and genome editing

Kishi Noriyuki, Okano Hideyuki

Genome Editing in Neurosciences    73 - 81 2017年

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<p>The common marmoset (Callithrix jacchus) is a small New World non-human primate indigenous to northeastern Brazil. This species has been attracting the attention of biomedical researchers and neuroscientists for its ease of handling and colony maintenance, unique behavioral characteristics, and several human-like traits, such as enriched social vocal communication and strong relationships between parents and offspring. Its high reproductive efficiency makes it particularly amenable for use in the development of transgenic and genome editing technologies in a non-human primate model. Our group has recently generated transgenic marmosets with germ line transmission, opening new avenues in primate research. In this chapter, we describe recent advances in neuroscience and disease research using common marmosets, and we outline potential uses of genome editing in non-human primates toward the development of knock-in/knock-out marmosets.</p>

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Astrocyte-mediated infantile-onset leukoencephalopathy mouse model

583) Sugio S, Tohyama K, Oku S, Fujiyoshi K, Yoshimura T, Hikishima K, Yano R, Fukuda T, Nakamura M, Okano H, Watanabe M, Fukata M, Ikenaka K, F Tanaka K

GLIA 65(1)   150 - 168 2017年

Technical feasibility of visualizing myenteric plexus using confocal laser endomicroscopy

591) Kobayashi M, Sumiyama K, Shimojima N, Shimojima N, Ieiri S, Okano H, Kamba S, Fujimura T, Hirobe S, Kuroda T, Takahashi-Fujigasaki J

J Gastroenterol Hepatol 2017年

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Enhanced functional recovery from spinal cord injury in aged mice after stem cell transplantation through HGF induction

593) Takano M, Kawabata S, Shibata S, Yasuda A, Nori S, Tsuji O, Iwanami A, Kohyama J, Ebise H, Horiuchi K, Okano H*, Nakamura M*

Stem Cell Reports 2017年