Naomi Nakagata

Kumamoto University, Kumamoto, Kumamoto Prefecture, Japan

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Publications (142)490.94 Total impact

  • Current Protocol in Mouse Biology. 12/2014; 4:205-227.
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    ABSTRACT: SNiemann-Pick disease type C (NPC) is a lysosomal storage disease characterized by abnormal accumulation of free cholesterol and glycolipids. Here, we established induced pluripotent stem cell (iPSC) lines from NPC patients. Hepatocyte-like cells (HLCs) and neural progenitors derived from the iPSC lines accumulated cholesterol and displayed impaired autophagy and ATP production. A molecular signature related to lipid metabolism was also impaired in the NPC-iPSC-derived HLCs. These findings indicate that iPSC-derived cells can phenocopy human NPC. We also newly found that 2-hydroxypropyl-γ-cyclodextrin (HPGCD) could reduce the cholesterol accumulation and restore the functional and molecular abnormalities in the NPC patient-derived cells, and do so more effectively than 2-hydroxypropyl-β-cyclodextrin treatment. In addition, NPC model mice showed an improved liver status and prolonged survival with HPGCDs. Thus, iPSC lines derived from patient cells are powerful tools to study cellular models of NPC, and HPGCD is a potential new drug candidate for future treatment of this disease. This article is protected by copyright. All rights reserved.
    Stem Cells 12/2014; · 7.70 Impact Factor
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    ABSTRACT: Masculinization of external genitalia is an essential process in the formation of the male reproductive system. Prominent characteristics of this masculinization are the organ size and the sexual differentiation of the urethra. Although androgen is a pivotal inducer of the masculinization, the regulatory mechanism under the control of androgen is still unknown. Here, we address this longstanding question about how androgen induces masculinization of the embryonic external genitalia through the identification of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb) gene. Mafb is expressed prominently in the mesenchyme of male genital tubercle (GT), the anlage of external genitalia. MAFB expression is rarely detected in the mesenchyme of female GTs. However, exposure to exogenous androgen induces its mesenchymal expression in female GTs. Furthermore, MAFB expression is prominently down-regulated in male GTs of androgen receptor (Ar) KO mice, indicating that AR signaling is necessary for its expression. It is revealed that Mafb KO male GTs exhibit defective embryonic urethral formation, giving insight into the common human congenital anomaly hypospadias. However, the size of Mafb KO male GTs is similar with that of wild-type males. Moreover, androgen treatment fails to induce urethral masculinization of the GTs in Mafb KO mice. The current results provide evidence that Mafb is an androgen-inducible, sexually dimorphic regulator of embryonic urethral masculinization.
    Proceedings of the National Academy of Sciences of the United States of America. 10/2014;
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    ABSTRACT: The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS) cell-derived macrophage-like cells for Alzheimer’s disease (AD). In previous studies, we established the technology to generate macrophage-like myeloid lineage cells with proliferating capacity from human iPS cells, and we designated the cells iPS-ML. iPS-ML reduced the level of Aβ added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aβ. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aβ. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2), which is a protease with Aβ-degrading activity. In vitro, expression of NEP2 but not anti-Aβ scFv enhanced the effect to reduce the level of soluble Aβ oligomer in the culture medium and to alleviate the neurotoxicity of Aβ. To analyze the effect of iPS-ML expressing NEP2 (iPS-ML/NEP2) in vivo, we intracerebrally administered the iPS-ML/NEP2 to 5XFAD mice, which is a mouse model of AD. We observed significant reduction in the level of Aβ in the brain interstitial fluid following administration of iPS-ML/NEP2. These results suggested that iPS-ML/NEP2 may be a potential therapeutic agent in the treatment of AD.
    Stem Cell Research. 10/2014;
  • Current Protocols in Mouse Biology. 09/2014; 4:85-104.
  • Current Protocols in Mouse Biology. 06/2014; 4:67-83.
  • Current Protocols in Mouse Biology. 06/2014; 4:47-65.
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    ABSTRACT: Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.
    Development 05/2014; 141(10):2157-64. · 6.60 Impact Factor
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    ABSTRACT: The BC (bulbocavernosus) is a sexually dimorphic muscle observed only in males. ARKO (Androgen receptor knock out) mouse studies show the loss of BC formation. This suggests that androgen signaling plays a vital role in its development. Androgen has been known to induce muscle hypertrophy through satellite cell activation and myonuclei accretion during muscle regeneration and growth. Whether the same mechanism is present during embryonic development is not yet elucidated. To identify the mechanism of sexual dimorphism during BC development, the timing of morphological differences was first established. It was revealed that the BC was morphologically different between male and female mice at E16.5. Differences in the myogenic process were detected at E15.5. The male BC possesses a higher number of proliferating undifferentiated myoblasts. To identify the role of androgen signalling in this process, muscle-specific androgen receptor (AR) mutation was introduced which resulted in no observable phenotypes. Hence, the expression of AR in the BC was examined and found that the AR did not colocalize with any muscle markers such as MyoD, Myog, and Pax7. It was revealed that the mesenchyme surrounding the BC expressed AR and the BC started to express AR at E15.5. AR mutation on the non-myocytic cells utilizing Sall1 (spalt-like transcription factor 1) Cre driver mouse was performed which resulted in defective BC formation. It was revealed that the number of proliferating undifferentiated myoblasts was reduced in the SallAR (Sall1 Cre:AR(L-/Y)) mutant embryos and the adult mutants were devoid of BC. The transition of myoblasts from proliferation to differentiation is mediated by Cdk-inhibitors. An increased expression of p21 was observed in the BC myoblast of the SallAR mutant and WT female. Altogether, this study suggets that the non-myocytic AR may paracrinely regulate the proliferation of myoblast possibly through inhibiting p21 expression in myoblasts of the BC.
    Endocrinology 04/2014; · 4.72 Impact Factor
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    ABSTRACT: Prostate is a male sex accessory organ. The prostatic epithelia consist primarily of basal and luminal cells that differentiate from embryonic urogenital sinus epithelia. Prostate tumors are believed to originate in the basal and luminal cells. However, factors that promote normal epithelial differentiation have not been well elucidated, particularly for bone morphogenetic protein (Bmp) signaling. This study shows that Bmp signaling prominently increases during prostatic differentiation in the luminal epithelia, which is monitored by the expression of pSmad1/5/8. To elucidate the mechanism of epithelial differentiation and the function of Bmp signaling during prostatic development, conditional male mutant mouse analysis for the epithelial-specific Bmp receptor 1a (Bmpr1a) was performed. We demonstrate that Bmp signaling is indispensable for luminal cell maturation, which regulates basal cell proliferation. Expression of the prostatic epithelial regulatory gene Nkx3.1 was significantly reduced in the Bmpr1a mutants. These results indicate that Bmp signaling is a key factor for prostatic epithelial differentiation, possibly by controlling the prostatic regulatory gene Nkx3.1.
    Endocrinology 04/2014; · 4.72 Impact Factor
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    ABSTRACT: The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. β-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of β-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the β-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (Shh(CreERT2)). Both β-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the β-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the β-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The Shh(CreERT2/+); β-catenin(flox(ex3)/+); BmprIA(flox/-) mutants displayed partial restoration of URS elongation compared with the β-catenin GOF mutants. These results indicate that some ARM phenotypes in the β-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal β-catenin signaling to the ARM phenotypes. These results are considered to shed light on the pathogenic mechanisms of human ARMs.Cell Death and Differentiation advance online publication, 14 March 2014; doi:10.1038/cdd.2014.21.
    Cell death and differentiation 03/2014; · 8.24 Impact Factor
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    ABSTRACT: Sperm cryopreservation has been widely adopted for maintenance of the genetically engineered mouse (GEM). The cryopreserved sperm are being exchanged among many institutes worldwide. However, the recipients are not always able to obtain high fertilization rates with the frozen sperm shipped from senders. In this study, we cryopreserved mouse sperm via various methods and performed in vitro fertilization (IVF) in which the combination of methyl-beta-cyclodextrin for sperm preincubation and reduced glutathione for insemination was used (the MBCD-GSH IVF). In addition, frozen sperm sent from the Jackson Laboratory (USA) were thawed and used for IVF in the same manner. The fertilization rates of both the sperm cryopreserved via the methods applied in some countries and the cryopreserved GEM sperm improved when used with the MBCD-GSH IVF method. Therefore, we strongly believe that the MBCD-GSH IVF method brings about relatively high fertilization rates with any strain of frozen mouse sperm.
    Journal of Reproduction and Development 02/2014; · 1.76 Impact Factor
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    ABSTRACT: Gene trapping in embryonic stem (ES) cells is a proven method for large-scale random insertional mutagenesis in the mouse genome. We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox-mediated recombination. We isolated trap clones, analyzed trapped genes, and constructed the database for Exchangeable Gene Trap Clones (EGTC) [http://egtc.jp]. The number of registered ES cell lines was 1162 on 31 August 2013. We also established 454 mouse lines from trap ES clones and deposited them in the mouse embryo bank at the Center for Animal Resources and Development, Kumamoto University, Japan. The EGTC database is the most extensive academic resource for gene-trap mouse lines. Because we used a promoter-trap strategy, all trapped genes were expressed in ES cells. To understand the general characteristics of the trapped genes in the EGTC library, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analysis and found that the EGTC ES clones covered a broad range of pathways. We also used Gene Ontology (GO) classification data provided by Mouse Genome Informatics (MGI) to compare the functional distribution of genes in each GO term between trapped genes in the EGTC mouse lines and total genes annotated in MGI. We found the functional distributions for the trapped genes in the EGTC mouse lines and for the RefSeq genes for the whole mouse genome were similar, indicating that the EGTC mouse lines had trapped a wide range of mouse genes.
    Embryologia 01/2014; · 2.21 Impact Factor
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    ABSTRACT: Hydroxypropyl-β-cyclodextrin (HPBCD) is an attractive drug candidate against Niemann–Pick Type C (NPC) disease. However, the safety of HPBCD treatment for NPC patients remains to be elucidated. In this study, we examined the acute toxicity of HPBCD in Npc1-deficient mice. When treated with HPBCD (20,000 mg/kg, subcutaneously), over half of the wild-type (Npc1+/+) or Npc1+/− mice died by 72 h after the injection. In contrast, all of the Npc1−/− mice survived.Marked pathophysiological changes, such as an elevation in serumtransaminase and creatinine levels, hepatocellular necrosis, renal tubular damage, interstitial thickening, and hemorrhages in lungs, were induced by the HPBCD treatment in Npc1+/+ or Npc1+/− mice. However, these pathophysiological changes were significantly alleviated in Npc1−/− mice. In addition, in vitro analysis showed that the Npc1 gene deficiency and treatment with U18666A, an Npc1 inhibitor, remarkably attenuated the cytotoxicity of HPBCD in Chinese hamster ovary cells. These results suggest that the NPC1 genotype exacerbates the cytotoxicity of HPBCD and Npc1−/− mice have substantial resistance to the lethality and the organ injury induced by HPBCD injection compared with Npc1+/+ or Npc1+/− mice.We suggest that the Npc1 genotype should be considered in the safety evaluation of HPBCD using experimental animals and cells.
    Molecular Genetics and Metabolism Reports. 01/2014; 1:19-30.
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    ABSTRACT: Hyaluronidase is generally used to remove cumulus cells from mouse oocytes before oocyte cryopreservation, intracytoplasmic sperm injection or DNA injection. In general, use of cumulus-free mouse oocytes decreases in vitro fertilizing ability compared with cumulus-surrounded oocytes. The effect of hyaluronidase exposure on the quality of mouse oocytes is not fully understood. Here, we investigated the effect of hyaluronidase exposure time on the fertilization rate of fresh and vitrified mouse oocytes and their subsequent developmental ability in vitro. We found that the fertilization rate decreased with hyaluronidase treatments. This reduction in the fertilization rate following treatment with hyaluronidase was fully reversed by removal of the zona pellucida. In addition, oocytes treated with hyaluronidase for 5 min or longer had a reduced capacity to develop to the morula and blastocyst stage. The survival, fertilization, and developmental rates of vitrified-warmed oocytes were also reduced by longer exposure to hyaluronidase. In conclusion, these results suggest that prolonged exposure to hyaluronidase decreases the quality of mouse oocytes and shorter hyaluronidase treatment times may help achieve a stable and high fertilization rate in fresh and cryopreserved oocytes.
    Journal of Reproduction and Development 01/2014; · 1.76 Impact Factor
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    ABSTRACT: Reproductive engineering techniques, such as in vitro fertilization (IVF) and cryopreservation of embryos or spermatozoa, are essential for preservation, reproduction, and transportation of genetically engineered mice. However, it has not yet been elucidated whether these techniques can be applied for the generation of genome-edited mice using engineered nucleases such as transcription activator-like effector nucleases (TALENs). Here, we demonstrate the usefulness of frozen oocytes fertilized in vitro using frozen sperm for TALEN-mediated genome editing in mice. We examined side-by-side comparisons concerning sperm (fresh vs. frozen), fertilization method (mating vs. IVF), and fertilized oocytes (fresh vs. frozen) for the source of oocytes used for TALEN injection; we found that fertilized oocytes created under all tested conditions were applicable for TALEN-mediated mutagenesis. In addition, we investigated whether the ages in weeks of parental female mice can affect the efficiency of gene modification, by comparing 5-week-old and 8-12-week-old mice as the source of oocytes used for TALEN injection. The genome editing efficiency of an endogenous gene was consistently 95-100% when either 5-week-old or 8-12-week-old mice were used with or without freezing the oocytes. Thus, our report describes the availability of freeze-thawed oocytes and oocytes from female mice at various weeks of age for TALEN-mediated genome editing, thus boosting the convenience of such innovative gene targeting strategies.
    Experimental Animals 01/2014; 63(3):349-355. · 1.46 Impact Factor
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    ABSTRACT: During organogenesis, Sonic hedgehog (Shh) possesses dual functions: Shh emanating from midline structures regulates the positioning of bilateral structures at early stages, whereas organ-specific Shh locally regulates organ morphogenesis at later stages. The mesonephros is a transient embryonic kidney in amniote, whereas it becomes definitive adult kidney in some anamniotes. Thus, elucidating the regulation of mesonephros formation has important implications for our understanding of kidney development and evolution. In Shh knockout (KO) mutant mice, the mesonephros was displaced towards the midline and ectopic mesonephric tubules (MTs) were present in the caudal mesonephros. Mesonephros-specific ablation of Shh in Hoxb7-Cre;Shh(flox/-) and Sall1(CreERT2/+);Shh(flox/-) mice embryos indicated that Shh expressed in the mesonephros was not required for either the development of the mesonephros or the differentiation of the male reproductive tract. Moreover, stage-specific ablation of Shh in Shh(CreERT2/flox) mice showed that notochord- and/or floor plate-derived Shh were essential for the regulation of the number and position of MTs. Lineage analysis of hedgehog (Hh)-responsive cells, and analysis of gene expression in Shh KO embryos suggested that Shh regulated nephrogenic gene expression indirectly, possibly through effects on the paraxial mesoderm. These data demonstrate the essential role of midline-derived Shh in local tissue morphogenesis and differentiation.
    Developmental Biology 12/2013; · 3.87 Impact Factor
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    ABSTRACT: Cell replacement therapy for diabetes mellitus requires cost-effective generation of high-quality, insulin-producing, pancreatic β cells from pluripotent stem cells. Development of this technique has been hampered by a lack of knowledge of the molecular mechanisms underlying β-cell differentiation. The present study identified reserpine and tetrabenazine (TBZ), both vesicular monoamine transporter 2 (VMAT2) inhibitors, as promoters of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Neurog3 (referred to henceforth as Ngn3)-positive endocrine precursors. VMAT2-controlled monoamines, such as dopamine, histamine and serotonin, negatively regulated β-cell differentiation. Reserpine or TBZ acted additively with dibutyryl adenosine 3',5'-cyclic AMP, a cell-permeable cAMP analog, to potentiate differentiation of embryonic stem (ES) cells into β cells that exhibited glucose-stimulated insulin secretion. When ES cell-derived β cells were transplanted into AKITA diabetic mice, the cells reversed hyperglycemia. Our protocol provides a basis for the understanding of β-cell differentiation and its application to a cost-effective production of functional β cells for cell therapy.
    Nature Chemical Biology 12/2013; · 12.95 Impact Factor
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    ABSTRACT: Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48 hours maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72 hours, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.
    Cryobiology 11/2013; · 2.14 Impact Factor
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    ABSTRACT: Although several syndromes include abnormalities of both the ventral body wall and external genitalia, the developmental bases of this correlation are largely unknown. Naturally occurring mutations in Aristaless-like 4 (Alx4, Strong's luxoid: Alx4(Lst)) have ventral body wall and pelvic girdle abnormalities. We sought to determine whether the development of the genital tubercle (GT) and its derivatives, the external genitalia, is affected by this mutation. We thus performed genetic and tissue labeling analyses in mutant mice. Alx4(Lst/Lst) mutants displayed hypoplasia of the dorsal GT and reduced expression of Fibronectin. We analyzed cell migration during GT formation by tissue labeling experiments and discovered that the cells located in the proximal segment of the umbilical cord (infra-umbilical mesenchyme) migrate toward the dorsal part of the GT. The Alx4(Lst/Lst) mutants also displayed augmented expression of Hh signal-related genes. Hence, we analyzed a series of combinatorial mutants for Alx4, Sonic hedgehog (Shh) and GLI-Kruppel family member 3 (Gli3). These phenotype-genotype analyses suggested a genetic interaction between Alx4 and Hh signaling during GT formation. Moreover, Hh gain-of-function mutants phenocopied some of these phenotypes. These observations reveal novel information regarding the pathogenic mechanisms of syndromic lower ventral body malformations, which are largely unknown.European Journal of Human Genetics advance online publication, 14 August 2013; doi:10.1038/ejhg.2013.160.
    European journal of human genetics: EJHG 08/2013; · 3.56 Impact Factor

Publication Stats

2k Citations
490.94 Total Impact Points

Institutions

  • 2000–2014
    • Kumamoto University
      • • Department of Reproductive Engineering
      • • Department of Organ Formation
      • • Department of Developmental Genetics
      • • Department of Diagnostic Medicine
      • • Center for Animal Resources and Development
      Kumamoto, Kumamoto Prefecture, Japan
  • 2012
    • Wakayama Medical University
      Wakayama, Wakayama, Japan
  • 2007–2011
    • Nagoya City University
      • Graduate School of Medical Sciences
      Nagoya, Aichi, Japan
  • 2003–2005
    • Keio University
      • School of Medicine
      Tokyo, Tokyo-to, Japan
    • Centre for Water Resources Development and Management
      Kunnamangalam, Kerala, India
  • 2004
    • Azabu University
      • School of Veterinary Medicine
      Sagamihara, Kanagawa-ken, Japan
  • 1997–2003
    • The University of Tokyo
      • • Faculty and Graduate School of Agriculture and Life Sceince
      • • Institute of Medical Science
      Tokyo, Tokyo-to, Japan