Researchers Database

hara hiromasa

    DivisionofRegenerativeMedicine,CenterformolecularMedicine Assistant Professor
Last Updated :2021/12/04

Researcher Information

J-Global ID

Research Areas

  • Life sciences / Genomics
  • Life sciences / Laboratory animal science

Academic & Professional Experience

  • 2020/04 - Today  Jichi Medical University分子病態治療研究センター 再生医学研究部講師
  • 2016/03 - 2020/03  Jichi Medical University分子病態治療研究センター 再生医学研究部助教
  • 2015/04 - 2016/02  National Institutes of Natural Sciences行動・代謝分子解析センター 遺伝子改変動物作製室NIPSリサーチフェロー
  • 2013/04 - 2015/03  日本学術振興会特別研究員

Education

  • 2012/04 - 2014/09  信州大学院  博士課程
  • 2010/04 - 2012/03  信州大学院  修士課程
  • 2006/04 - 2010/03  Shinshu University  Faculty of Textile Science and Technology  Division of Applied Biology

Published Papers

  • Suvd Byambaa, Hideki Uosaki, Tsukasa Ohmori, Hiromasa Hara, Hitoshi Endo, Osamu Nureki, Yutaka Hanazono
    Molecular therapy. Methods & clinical development 20 451 - 462 2021/03 
    We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.
  • Tatsuya Anzai, Hiromasa Hara, Nawin Chanthra, Taketaro Sadahiro, Masaki Ieda, Yutaka Hanazono, Hideki Uosaki
    Methods in molecular biology (Clifton, N.J.) 2320 247 - 259 2021 
    A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.
  • Tomoyuki Abe, Hideki Uosaki, Hiroaki Shibata, Hiromasa Hara, Borjigin Sarentonglaga, Yoshikazu Nagao, Yutaka Hanazono
    Experimental Hematology 0301-472X 2021 
    © 2021 ISEH – Society for Hematology and Stem Cells We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)–derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.
  • Tomoyuki Abe, Hideki Uosaki, Hiroaki Shibata, Hiromasa Hara, Borjigin Sarentonglaga, Yoshikazu Nagao, Yutaka Hanazono
    Experimental hematology 2021/01 
    We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.
  • Byambaa S, Uosaki H, Hara H, Nagao Y, Abe T, Shibata H, Nureki O, Ohmori T, Hanazono Y
    Experimental animals 69 (2) 189 - 198 1341-1357 2019/12 [Refereed][Not invited]
     
    X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.
  • 阿部 朋行, 柴田 宏昭, 魚崎 英毅, 原 弘真, ボラジギン・サラントラガ, 長尾 慶和, 花園 豊
    Organ Biology (一社)日本臓器保存生物医学会 26 (3) 87 - 87 1340-5152 2019/10
  • 原 弘真, 伊藤 拓哉, 菱川 修司, 中野 和明, 阿部 朋行, 柴田 宏昭, 魚崎 英毅, 渡邊 將人, 國田 智, 長嶋 比呂志, 花園 豊
    Organ Biology (一社)日本臓器保存生物医学会 26 (3) 89 - 89 1340-5152 2019/10
  • 原 弘真, 菱川 修司, 伊藤 拓哉, 阿部 朋行, 柴田 宏昭, 魚崎 英毅, 國田 智, 花園 豊
    Organ Biology (一社)日本臓器保存生物医学会 26 (3) 121 - 121 1340-5152 2019/10
  • 和食の健康維持・増進効果の機序解明を目指した研究
    原 弘真, 永山 学, 須田 亙, 柴田 宏昭, 菱川 修司, 國田 智, 阿部 朋行, 魚崎 英毅, 新 幸二, 本田 賢也, 花園 豊
    自治医科大学紀要 (学)自治医科大学 41 92 - 92 1881-252X 2019/03
  • Goto T, Hara H, Sanbo M, Masaki H, Sato H, Yamaguchi T, Hochi S, Kobayashi T, Nakauchi H, Hirabayashi M
    Nature communications 10 (1) 451  2019/02 [Refereed][Not invited]
  • Yamaguchi T, Sato H, Kobayashi T, Kato-Itoh M, Goto T, Hara H, Mizuno N, Yanagida A, Umino A, Hamanaka S, Suchy F, Masaki H, Ota Y, Hirabayashi M, Nakauchi H
    Scientific reports 8 (1) 15289 - 15289 2018/10 [Refereed][Not invited]
     
    To study development of the conceptus in xenogeneic environments, we assessed interspecies chimera formation as well as tetraploid complementation between mouse and rat. Overall contribution of donor PSC-derived cells was lower in interspecies chimeras than in intraspecies chimeras, and high donor chimerism was associated with anomalies or embryonic death. Organ to organ variation in donor chimerism was greater in interspecies chimeras than in intraspecies chimeras, suggesting species-specific affinity differences among interacting molecules necessary for organogenesis. In interspecies tetraploid complementation, embryo development was near normal until the stage of placental formation, after which no embryos survived.
  • Hiromasa Hara, Hiroaki Shibata, Kazuaki Nakano, Tomoyuki Abe, Hideki Uosaki, Takahiro Ohnuki, Shuji Hishikawa, Satoshi Kunita, Masahito Watanabe, Osamu Nureki, Hiroshi Nagashima, Yutaka Hanazono
    Experimental Animals 67 (2) 139 - 146 1341-1357 2018 [Refereed][Not invited]
     
    Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20–100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=−0.97, P< 0.05). Therefore, it is critical to deliver piglets within 5 min to achieve a high resuscitation rate (82% estimated from regression analysis). In a fifth trial with an IL2RG+/− pig, four piglets were delivered within 4.2 min of uterus excision and three were alive (75%). One of the live born piglets was genotypically and phenotypically determined to be X-SCID and was reared for 12 weeks. The X-SCID piglet was free from both bacteria and fungi at all time points tested by microbial culture and grew without any abnormal signs or symptoms. This study showed successful production and rearing of germ-free pigs, enabling experiments involving long-term follow-up of X-SCID pigs.
  • Masumi Hirabayashi, Hiromasa Hara, Teppei Goto, Akiko Takizawa, Melinda R. Dwinell, Takahiro Yamanaka, Shinichi Hochi, Hiromitsu Nakauchi
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 63 (6) 611 - 616 0916-8818 2017/12 [Refereed][Not invited]
     
    The present study was conducted to establish haploid embryonic stem (ES) cell lines using fluorescent marker carrying rats. In the first series, 7 ES cell lines were established from 26 androgenetic haploid blastocysts. However, only 1 ES cell line (ahES-2) was found to contain haploid cells (1n = 20 + X) by fluorescence-activated cell sorting (FAGS) and karyotypic analyses. No chimeras were detected among the 10 fetuses and 41 offspring derived from blastocyst injection with the FACS-purified haploid cells. In the second series, 2 ES cell lines containing haploid cells (13% in phES-1 and 1% in phES-2) were established from 2 parthenogenetic haploid blastocysts. Only the phES-2 cell population was purified by repeated FAGS to obtain 33% haploid cells. Following blastocyst injection with the FAGS-purified haploid cells, no chimera was observed among the 11 fetuses; however, 1 chimeric male was found among the 47 offspring. Although haploid rat ES cell lines can be established from both blastocyst sources, FAGS purification may be necessary for maintenance and chimera production.
  • Tomoyuki Yamaguchi, Hideyuki Sato, Megumi Kato-Itoh, Teppei Goto, Hiromasa Hara, Makoto Sanbo, Naoaki Mizuno, Toshihiro Kobayashi, Ayaka Yanagida, Ayumi Umino, Yasunori Ota, Sanae Hamanaka, Hideki Masaki, Sheikh Tamir Rashid, Masumi Hirabayashi, Hiromitsu Nakauchi
    NATURE 542 (7640) 191 - 196 0028-0836 2017/02 [Refereed][Not invited]
     
    Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse-rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.
  • Teppei Goto, Hiromasa Hara, Hiromitsu Nakauchi, Shinichi Hochi, Masumi Hirabayashi
    TRANSGENIC RESEARCH 25 (4) 533 - 544 0962-8819 2016/08 [Refereed][Not invited]
     
    The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.
  • Hiromasa Hara, Teppei Goto, Akiko Takizawa, Makoto Sanbo, Howard J. Jacob, Toshihiro Kobayashi, Hiromitsu Nakauchi, Shinichi Hochi, Masumi Hirabayashi
    CELLULAR REPROGRAMMING 18 (2) 108 - 115 2152-4971 2016/04 [Refereed][Not invited]
     
    Pronucleus-like vesicle formation following premature chromosome condensation (PCC) of the donor cell nucleus is the key event for successful generation of cloned rodents by nuclear transplantation (NT). However in rat cloning, this change is difficult to induce in enucleated recipient oocytes because of their inability to maintain maturation-promoting factor levels. In this study, intact oocytes retrieved from nuclear-visualized H2B-tdTomato knock-in rats were injected with Venus-labeled cell nuclei. Because the incidence of PCC under MG-132 treatment significantly increased with the culture period (0%, 10.8%, 36.8%, and 87.5% at 0, 0.5, 1, and 2 h postinjection, respectively), the metaphase plate of the oocyte was removed 1-2 h after the nuclear injection. The NT-derived rat zygotes (n = 748) were activated with ionomycin/cycloheximide and transferred into temporal host mothers, resulting in the harvest of three blastocysts (0.4%) with Venus fluorescence. Two blastocysts were examined for their potential to commit to NT-derived embryonic stem cells (ntESCs). One ntESC line was established successfully and found to be competent in terms of karyotype, stem cell marker expression, and pluripotency. In conclusion, time-lagged enucleation of visualized oocyte nuclei allows the PCC incidence of donor nuclei and generation of NT blastocysts, and the blastocysts can commit to germline-competent ntESCs.
  • H. Hara, M. Tagiri, M. Hirabayashi, S. Hochi
    ZYGOTE 24 (1) 115 - 120 0967-1994 2016/02 [Refereed][Not invited]
     
    We have recently reported that multiple aster formation after in vitro fertilization (IVF) was one of the factors that negatively affected the developmental competence of vitrified-warmed bovine matured oocytes, and that short-term culture of the post-warm oocytes with an inhibitor of Rho-associated coiled-coil kinase (ROCK) suppressed the multiple aster formation and improved the blastocyst yield. The present study was conducted to investigate whether increased multiple aster formation following IVF was involved in impaired developmental competence of stored ovary-derived bovine oocytes. Oocytes retrieved from 1-day stored ovaries had lower developmental potential to day 8 blastocysts when compared with those from fresh ovaries (37 versus 63%). Immunostaining of alpha-tubulin 10 h post-IVF revealed that a higher incidence of multiple aster formation occurred in oocytes retrieved from stored ovaries than from fresh ovaries (31 versus 15%). Treatment of post-in vitro maturated (post-IVM) oocytes with ROCK inhibitor for 2 h significantly suppressed the incidence of multiple aster formation (10 versus 32% in the control group). However, the suppression effect of ROCK inhibitor on multiple aster formation in IVM/IVF oocytes did not improve blastocyst yield from stored ovary-derived oocytes (41 versus 37% in the control group). These results suggested that the higher incidence of multiple aster formation by bovine ovary storage was not responsible for the decreased developmental competence of IVF oocytes.
  • Teppei Goto, Toshihiro Kobayashi, Hiromasa Hara, Makoto Sanbo, Shinichi Hochi, Hiromitsu Nakauchi, Masumi Hirabayashi
    MOLECULAR REPRODUCTION AND DEVELOPMENT 82 (12) 916 - 917 1040-452X 2015/12 [Refereed][Not invited]
  • Ikuko Yashiro, Miho Tagiri, Hayato Ogawa, Kazuya Tashima, Seiji Takashima, Hiromasa Hara, Masumi Hirabayashi, Shinichi Hochi
    REPRODUCTION 149 (4) 347 - 355 1470-1626 2015/04 [Refereed][Not invited]
     
    The objective of this study was to investigate whether developmental competence of vitrified-warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either L-ascorbic acid or alpha-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to a-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1-36.3% vs 19.2-25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between alpha-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in a-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with alpha-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that alpha-tocopherol treatment of vitrified-warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.
  • H. Hara, I. Yamane, I. Noto, N. Kagawa, M. Kuwayama, M. Hirabayashi, S. Hochi
    ZYGOTE 22 (4) 476 - 482 0967-1994 2014/11 [Refereed][Not invited]
     
    Although vitrification is a useful technique for preservation of bovine oocytes, the yield of blastocysts derived from the vitrified oocytes is still low. We have recently reported a new type of cryoinjury, multiple aster formation, by which pronuclear migration and development of vitrified-warmed and in vitro-fertilized bovine oocytes are impaired. The aim of the present study was to investigate the effect of glutathione (GSH) content of vitrified bovine oocytes on multiple aster formation and subsequent in vitro development. Treatment of bovine cumulus-oocyte complexes with beta-mercaptoethanol (beta ME) and L-cysteine (Cys) during in vitro maturation resulted in 2.5-fold higher GSH content not only in fresh control but also in vitrified-warmed oocytes. The percentage of normally fertilized zygotes exhibiting sperm aster(s) was >95% in all four groups (with or without beta ME/Cys x fresh control or vitrified). The frequency of multiple aster formation in vitrified oocytes (three-fold higher than that in fresh control oocytes) was not affected by the increased level of intracellular GSH with beta ME/Cys. Consequently, the migration and development of pronuclei as well as the yield of blastocysts from vitrified-warmed oocytes (17 versus 41%) were not improved. In addition, there was no effect of increased GSH level on the yield of blastocysts in fresh control groups.
  • Hiromasa Hara, Miho Tagiri, In-Sul Hwang, Masato Takahashi, Masumi Hirabayashi, Shinichi Hochi
    CRYOBIOLOGY 68 (3) 354 - 360 0011-2240 2014/06 [Refereed][Not invited]
     
    Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (T-g') during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the T-g' of conventional EGTA buffer (consisting of Tris-HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer T-g' was too low (-45.0 degrees C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of T-g' up to -27.7 degrees C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or -15 degrees C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: -30 degrees C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both T-g' of freeze-drying buffer and temperature during the drying phase. (C) 2014 Elsevier Inc. All rights reserved.
  • Masumi Hirabayashi, Teppei Goto, Chihiro Tamura, Makoto Sanbo, Hiromasa Hara, Shinichi Hochi
    Journal of Reproduction and Development 60 (1) 78 - 82 0916-8818 2014 [Refereed][Not invited]
     
    This study was designed to investigate whether supplementation of 2i medium with leukemia inhibitory factor (LIF) and/or forskolin would support establishment of germline-competent rat embryonic stem (ES) cell lines. Due to the higher likelihood of outgrowth rates, supplementation of forskolin with or without LIF contributed to the higher establishment efficiency of ES cell lines in the WDB strain. Germline transmission competency of the chimeric rats was not influenced by the profile of ES cell lines until their establishment. When the LIF/forskolin-supplemented 2i medium was used, the rat strain used as the blastocyst donor, such as the WI strain, was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, all lines were found to be germline-competent by a progeny test in chimeric rats. In conclusion, both LIF and forskolin are not essential but can play a beneficial role in the establishment of "genuine" rat ES cell lines. © 2014 by the Society for Reproduction and Development.
  • Masumi Hirabayashi, Teppei Goto, Chihiro Tamura, Makoto Sanbo, Hiromasa Hara, Megumi Kato-Itoh, Hideyuki Sato, Toshihiro Kobayashi, Hiromitsu Nakauchi, Shinichi Hochi
    STEM CELLS AND DEVELOPMENT 23 (2) 107 - 114 1547-3287 2014/01 [Refereed][Not invited]
     
    This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.
  • In-Sul Hwang, Hiromasa Hara, Hak-Jae Chung, Masumi Hirabayashi, Shinichi Hochi
    BIOLOGY OF REPRODUCTION 89 (2) 26  0006-3363 2013/08 [Refereed][Not invited]
     
    Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rhoassociated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 mu M Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.
  • H. Hara, I-S Hwang, N. Kagawa, M. Kuwayama, M. Hirabayashi, S. Hochi
    THERIOGENOLOGY 77 (5) 908 - 915 0093-691X 2012/03 [Refereed][Not invited]
     
    In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster. (C) 2012 Elsevier Inc. All rights reserved.
  • Hiromasa Hara, Hany Abdalla, Hiroshi Morita, Masashige Kuwayama, Masumi Hirabayashi, Shinichi Hochi
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 57 (3) 428 - 432 0916-8818 2011/06 [Refereed][Not invited]
     
    This study was designed to investigate whether freeze-dried (FD) bull spermatozoa maintained the function of the microtubule-organizing center (MTOC) after rehydration and intracytoplasmic sperm injection (ICSI). In a preliminary attempt, the cleavage and blastocyst formation rates in FD-ICSI zygotes (36 and 1%, respectively) were found to be considerably lower than those in control ICSI zygotes (67 and 21%, respectively) or in IVF zygotes (78 and 43%, respectively). An alkaline comet assay indicated that the DNA fragmentation index (length of comet tail x % DNA liberated) was not significantly different between fresh and FD spermatozoa. In the main experiment, formation of sperm-asters in the FD-ICSI oocytes 7 h postinsemination occurred at a similar rate when compared with the control ICSI oocytes (41 vs. 49%). Among the oocytes exhibiting sperm aster formation, the extent of microtubule network assembly was comparable between the FD-ICSI and control ICSI groups. However, the MTOC of the ICSI oocytes was not as functional as that of IVF oocytes in terms of the aster formation rate (97%) and the fluorescent intensity of the microtubule network (2.0 folds). These results suggest that the freeze-drying process per se had no adverse effect on maintaining the MTOC function in bull spermatozoa.
  • H. Abdalla, M. Shimoda, H. Hara, H. Morita, M. Kuwayama, M. Hirabayashi, S. Hochi
    THERIOGENOLOGY 74 (6) 1028 - 1035 0093-691X 2010/10 [Refereed][Not invited]
     
    The objective was to investigate the effects of developmental stage (fully-expanded or expanding blastocysts) and/or age (harvested on Days 7 or 8) on post-vitrification in vitro survival of bovine blastocysts derived from intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Post-warming survival (re-expansion of blastocoele within 24 h) of ICSI-derived fully-expanded blastocysts (80%) was similar to that of their IVF-derived counterparts (88%). However, the ability of ICSI-derived expanding blastocysts to survive vitrification procedures (61%) was lower than that of IVF-derived blastocysts (85%; P < 0.05), although the ICSI- and IVF-derived fresh blastocysts were of similar quality. The age of the blastocysts before vitrification did not affect cryotolerance for either ICSI-derived (73 and 59% for Days 7 and 8 embryos, respectively) or IVF-derived blastocysts (86% for both Days 7 and 8 embryos). At 24 h of post-warming culture, ICSI-derived blastocysts surviving vitrification contained a higher proportion of dead cells than their IVF-derived counterparts (5-13% vs. 2-4%; P < 0.05), but these proportions were not different from those of fresh control embryos. There was an adverse effect of vitrification on the ability of blastocysts to hatch within 72 h of culture only in IVF-derived Day 8 blastocysts (41 and 70% in vitrified and fresh control groups, respectively). In conclusion, the proportion of blastocysts that survived vitrification procedures was similar for ICSI- and IVF-derived bovine blastocysts if the former were cultured to the fully-expanded stage prior to vitrification, with no significant difference between embryos harvested on Day 7 versus Day 8. (C) 2010 Elsevier Inc. All rights reserved.
  • S. Hochi, H. Abdalla, H. Hara, M. Shimoda, H. Morita, M. Kuwayama, M. Hirabayashi
    THERIOGENOLOGY 73 (8) 1139 - 1145 0093-691X 2010/05 [Refereed][Not invited]
     
    Inhibition of Rho-associated kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium unproved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol. 15% DMSO and 0 5 M sucrose When post-warm blastocysts were cultured in inSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved (P < 0 05) by the addition of 10 mu M Y-27632 (94 9 +/- 2 4%, mean +/- SEM) compared to a control (78 0 +/- 6 0%) Conversely. after 48 h of culture, there were no significant differences in hatching late (62.8 +/- 11 1 vs 59 6 +/- 9.4%) and mean total cell number (135 2 +/- 13 1 vs. 146 7 +/- 13 3) In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91 7 +/- 3 8 vs 54 7 +/- 8 9%. P < 0 05). with no difference in mean total cell number of blastocysts (230 0 +/- 23 0 vs 191 2 +/- 22 2, P = 0 23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2. or Day 4 (and remained present until Day 8). resulting in no improvement in blastocyst yield compared to a control group (7.5 +/- 2 1, 31 4 +/- 2 3, 36 2 +/- 3.2. and 28.6 +/- 6.9%. respectively) In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming (C) 2010 Elsevier Inc. All rights reserved

Conference Activities & Talks

  • 腸内細菌叢ヒト化ピッグ  [Not invited]
    原弘真
    自治医科大学先端医療技術開発センターシンポジウム2020  2020/02
  • PHENOTYPICAL CORRECTION OF X-SCID MICE AFTER CRISPR/CAS9-MEDIATED UNIVERSAL GENOME-EDITING THERAPY OF HEMATOPOIETIC STEM CELLS  [Not invited]
    Suvd Byambaa, Hideki Uosaki, Hiromasa Hara, Hiroaki Shibata, Tomoyuki Abe, Yasumitsu Nagao, Osamu Nureki, Tsukasa Ohmori, Yutaka Hanazono
    Frontiers in Genome Engineering 2019  2019/11
  • ESTABLISHMENT OF SIMULTANEOUS TRIPLE-REPORTER SYSTEM FOR HDR/NHEJ/DSB  [Not invited]
    Hiromasa Hara, Hideki Uosaki, Tomoyuki Abe, Hiroaki Shibata, Osamu Nureki, Yutaka Hanazono
    Frontiers in Genome Engineering 2019  2019/11
  • 大型動物胎仔を用いたヒト臓器再生技術:ヒツジで作るヒト造血幹細胞  [Not invited]
    阿部朋行, 柴田宏昭, 魚崎英毅, 原 弘真, ボラジギン・サラントラガ, 長尾慶和, 花園 豊
    第46回日本臓器保存生物医学会学術集会  2019/11
  • ピッグ同種輸血の試み  [Not invited]
    原弘真, 菱川修司, 伊藤拓哉, 阿部朋行, 柴田宏昭, 魚崎英毅, 國田智, 花園豊
    第46回日本臓器保存生物医学会学術集会  2019/11
  • SCIDピッグの長期飼育:再生医療・細胞治療評価系の確立をめざして  [Not invited]
    原弘真, 伊藤拓哉, 菱川修司, 中野和明, 阿部朋行, 柴田宏昭, 魚崎英毅, 渡邊將人, 國田智, 長嶋比呂志, 花園豊
    第46回日本臓器保存生物医学会学術集会  2019/11
  • 汎血球減少ピッグへの同種輸血  [Not invited]
    原弘真, 菱川修司, 伊藤拓哉, 阿部朋行, 柴田宏昭, 魚崎英毅, 國田智, 花園豊
    日本先進医工学ブタ研究会  2019/10
  • Phenotypical Correction of X-SCID Mice After CRISPR/Cas9-mediated Genome-Editing Therapy of Hematopoietic Stem Cells  [Not invited]
    Byambaa S, Uosaki H, Hara H, Shibata H, Abe T, Nagao Y, Nureki O, Ohmori T, Hanazono Y
    第25回日本遺伝子細胞治療学会学術集会  2019/07
  • Cure of X-SCID Mice After Ex Vivo Non-viral Genome-Editing Therapy of Hematopoietic Stem Cells  [Not invited]
    Byambaa S, Uosaki H, Hara H, Shibata H, Abe T, Hayakawa H, Nagao Y, Nureki O, Ohmori T, Hanazono Y
    the 21st American Society of Gene and Cell therapy  2019/05
  • Targeted Gene Integration in Hematopoietic Stem and Progenitor Cells with Cas9-RNP Method to Universally Correct X-SCID Mutations.  [Not invited]
    Byambaa S, Uosaki H, Hara H, Shibata H, Abe T, Nagao Y, Nureki O, Ohmori T, Hanazono Y
    第41回日本分子生物学会  2018/11  神奈川県横浜市  日本分子生物学会
  • 腸内細菌研究におけるピッグのモデル動物としての可能性  [Not invited]
    原弘真, 永山学, 須田亙, 魚崎英毅, 阿部朋行, 柴田宏昭, 菱川修司, 國田智, 新幸二, 本田賢也, 花園豊
    第6回先進医工学ブタ研究  2018/10  静岡県三島市  日本先進医工学ブタ研究会
  • Novel SCID mice generation by CRISPR/Cas9  [Not invited]
    Byambaa S, Uosaki H, Hara H, Shibata H, Abe T, Nagao Y, Nureki O, Ohmori T, Hanazono Y
    第65回日本実験動物学会  2018/05  富山県富山市  日本実験動物学会
  • ヒト腸内細菌定着ブタとヒトの腸内細菌叢の比較  [Not invited]
    原弘真, 永山学, 須田亙, 大貫貴広, 菱川修司, 柴田宏昭, 阿部朋行, 魚崎英毅, 國田智, 新幸二, 本田賢也, 花園豊
    第65回日本実験動物学会  2018/05  富山県富山市  日本実験動物学会
  • 新生ブタに対する麻酔管理  [Not invited]
    伊藤拓哉, 芝順太郎, 五十嵐孝, 五十嵐かほり, 半田紀子, 菱川修司, 柴田宏明, 魚崎英毅, 原弘真, 和久井亨, 宮澤和志, 栗田瑞希, 伊藤昌紀, 國田智, 竹内護, 花園豊
    第65回日本実験動物学会  2018/05  富山県富山市  日本実験動物学会
  • 異種移植系における同一ドナー由来リンパ球の影響  [Not invited]
    阿部朋行, 柴田宏昭, 原 弘真, 魚崎英毅, 大貫貴広, 竹内絢香, 原明日香, ボラジギン・サラントラガ, 長尾慶和, 花園 豊
    第20回日本異種移植研究会  2018/03  大阪  日本異種移植研究会
  • Generation of germfree pigs and their applications  [Not invited]
    Hara H, Hanazono Y
    The 14th Nikko International Symposium 2017  2017/10
  • ヒト腸内細菌定着ブタの作出と腸内細菌叢のメタ16S解析  [Not invited]
    原弘真, 永山学, 柴田宏昭, 須田亙, 大貫貴広, 菱川修司, 國田智, 阿部朋行, 新幸二, 本田賢也, 花園豊
    第5回先進医工学ブタ研究会  2017/10  静岡県三島市  日本先進医工学ブタ研究会
  • 無菌環境下におけるSCIDブタ長期飼育の試み  [Not invited]
    原弘真, 柴田宏昭, 中野和明, 阿部朋行, 魚崎英毅, 大貫貴広, 菱川修司, 國田智, 渡邊將人, 濡木理, 長嶋比呂志, 花園豊
    第5回先進医工学ブタ研究会  2017/10  静岡県三島市  日本先進医工学ブタ研究会
  • Generation of CD45-positive Hematopoietic Cells from Human iPS Cells in Vivo in Ovine Fetal Liver  [Not invited]
    Abe T, Shibata H, Uosaki H, Hara H, Ohnuki T, Byambaa S, Chanthra N, Sarentonglaga B, Fukumori R, Nagao Y, Hanazono Y
    International Society for Stem Cell Research 2017 Annual Meeting  2017/06  Boston, USA
  • ヒトiPS細胞由来造血細胞のヒツジ体内での長期生着  [Not invited]
    阿部朋行, 柴田宏昭, 魚崎英毅, 原弘真, 大貫貴広, スブド ビャンバー, ナーウィン ジャントラー, ボラジギン サラントラガ, 福森理加, 長尾慶和, 花園豊
    第64回日本実験動物学会  2017/05  福島県郡山市  日本実験動物学会
  • ブタの無菌的娩出・飼育技術の確立とSCIDブタ飼育の試み  [Not invited]
    原弘真, 柴田宏昭, 中野和明, 阿部朋行, 魚崎英毅, 大貫貴広, スブド ビャンバー, ナーウィン ジャントラー, 菱川修司, 國田智, 長嶋比呂志, 濡木理, 花園豊
    第64回日本実験動物学会  2017/05  福島県郡山市  日本実験動物学会
  • Resistance of Mouse Hematopoietic Stem and Progenitor Cells to the Genome-Editing with Cas9  [Not invited]
    Byambaa S, Uosaki H, Hara H, Abe T, Nagao Y, Nureki O, Ohmori T, Hanazono Y
    the 20th American Society of Gene and Cell Therapy  2017/05  Washington DC, USA
  • ブスルファンによる血小板減少モデルブタの作出  [Not invited]
    大貫貴広, 阿部朋行, 河野正太, 原弘真, 柴田宏昭, 菱川修司, 國田智, 花園豊
    第4回日本先進医工学ブタ研究会  2016/10  静岡県三島市  日本先進医工学ブタ研究会
  • 血小板減少モデルブタに輸注したヒト血小板の体内動態  [Not invited]
    阿部朋行, 大貫貴広, 河野正太, 原弘真, 柴田宏昭, 菱川修司, 國田智, 花園豊
    第4回日本先進医工学ブタ研究会  2016/10  静岡県三島市  日本先進医工学ブタ研究会
  • 無菌ブタの作出とその無菌的管理技術の確立  [Not invited]
    原弘真, 大貫貴広, 河野正太, 阿部朋行, 柴田宏昭, 中野和明, 長嶋比呂志, 菱川修司, 國田智, 花園豊
    第4回日本先進医工学ブタ研究会  2016/10  静岡県三島市  日本先進医工学ブタ研究会
  • The fates of infused human platelets in busulfan-induced thrombocytopenic pigs  [Not invited]
    Abe T, Ohnuki T, Hara H, Shibata H, Kono S, Hanazono Y
    第15回 自治医科大学シンポジウム  2016/09  栃木県下野市
  • Haploid ES cell lines derived from androgenetic and parthenogenetic rat blastocysts  [Not invited]
    Hirabayashi M, Hara H, Goto T, Takizawa A, Dwinell MR, Hochi S, Nakauchi H
    49th Annual Meeting of the Society for the Study of Reproduction  2016/07
  • Mouse thymus generated in Foxn1 mutant rats by blastocyst complementation  [Not invited]
    Goto T, Hara H, Hochi S, Nakauchi H, Hirabayashi M
    49th Annual Meeting of the Society for the Study of Reproduction  2016/07
  • 雄性発生胚ならびに雌性発生胚からの半数体ラットES細胞株の樹立  [Not invited]
    原弘真, 後藤哲平, 滝沢明子, DWINELL Melinda, 保地眞一, 中内啓光, 平林真澄
    第63回日本実験動物学会  2016/05
  • 腎発生関連遺伝子Sall1をノックアウトしたラットの表現型解析  [Not invited]
    後藤哲平, 原弘真, 保地眞一, 中内啓光, 平林真澄
    第63回日本実験動物学会  2016/05
  • 臓器再生に向けたCRISPR/Cas9法による腎臓欠損ラットの作出  [Not invited]
    後藤 哲平, 原 弘真, 保地 眞一, 平林 真澄
    第108回日本繁殖生物学会  2015/09
  • ドナー核注入後に時間をおいて除核する方法で作製したラットクローン胚盤胞  [Not invited]
    原弘真, 滝澤明子, 後藤哲平, 三宝誠, 小林俊寛, 中内啓光, 保地眞一, 平林真澄
    第108回日本繁殖生物学会  2015/09
  • Rat blastocysts resulting from somatic cell nuclear injection and time-lagged enucleation  [Not invited]
    Hara H, Goto T, Sanbo M, Kobayashi T, Nakauchi H, Hochi S, Hirabayashi M
    48th Annual Meeting of the Society for the Study of Reproduction  2015/06
  • CRISPR/Cas9法により作出したFoxN1遺伝子ノックアウトラットの解析  [Not invited]
    後藤哲平, 原弘真, 保地眞一, 平林真澄
    第62回日本実験動物学会  2015/05
  • 回復培養におけるα-トコフェロール処理はガラス化ウシ卵子の発生能を改善する  [Not invited]
    原弘真, 八代育子, 田切美穂, 平林真澄, 保地眞一
    第4回生理学研究所・名古屋大学医学系研究科合同シンポジウム  2014/11
  • 保存卵巣由来のウシ前核期卵における星状体形成と胚盤胞発生との関係  [Not invited]
    原弘真, 田切美穂, 平林真澄, 保地眞一
    第107回日本繁殖生物学会  2014/08
  • 保存卵巣由来のウシ前核期卵における星状体形成ならびにその後の胚盤胞発生に及ぼす短期ROCK阻害処理の影響  [Not invited]
    田切美穂, 原弘真, 黄仁偰, 八代育子, 平林真澄, 保地眞一
    第55回日本卵子学会  2014/05
  • ガラス化ウシ成熟卵母細胞の回復培養液におけるビタミンC及びEの添加効果  [Not invited]
    八代育子, 田切美穂, 能登一葉, 山根伊織, 原 弘真, 保地眞一
    第118回日本畜産学会  2014/03
  • Effect of cake collapse on integrity of freeze-dried bull spermatozoa  [Not invited]
    Hara H, Tagiri M, Hirabayashi M, Hochi S
    40th Annucal Conference of the International Embryo Transfer Society  2014/01
  • 凍結乾燥ウシ精子の発生能に及ぼすコラプスの影響  [Not invited]
    原弘真, 田切美穂, 平林真澄, 保地 眞一
    第106回日本繁殖生物学会  2013/09
  • Rescue of vitrified-warmed bovine oocytes by recovery culture in the presence of Rho-associated coiled-coil kinase (ROCK) inhibitor  [Not invited]
    黄仁偰, 八代育子, 原弘真, 平林真澄, 保地眞一
    第106回日本繁殖生物学会  2013/09
  • 成熟培養におけるROCK阻害が保存卵巣に由来するウシ卵母細胞の発生能に及ぼす影響  [Not invited]
    山根伊織, 原弘真, 平林真澄, 保地眞一
    第117回日本畜産学会  2013/09
  • 凍結乾燥ケーキにおけるコラプス形成と復水ウシ精子の正常性との関係  [Not invited]
    原弘真, 田切美穂, 平林真澄, 保地眞一
    第116回日本畜産学会  2013/03
  • ガラス化したウシ成熟卵母細胞の蘇生率は回復培養液へ添加したROCK阻害剤によって改善される  [Not invited]
    八代育子, 黄仁偰, 山根伊織, 能登一葉, 原弘真, 平林真澄, 保地眞一
    第116回日本畜産学会  2013/03
  • ガラス化・加温したウシ体外成熟卵子における細胞内グルタチオン濃度と精子星状体形成及び胚盤胞発生との関係  [Not invited]
    原弘真, 能登一葉, 山根伊織, 香川則子, 桑山正成, 平林真澄, 保地眞一
    第105回日本繁殖生物学会  2012/09
  • Apoptosis in bovine blastocysts derived from vitrified-warmed oocytes  [Not invited]
    Hwang IS, Hara H, Hochi S
    The 6th International Conference on Advanced Fiber/Textile Materials  2011/12
  • ガラス化・加温後に体外受精したウシ卵母細胞に多発した複数の星状体形成  [Not invited]
    原弘真, HWANG In-Sul, 桑山正成, 平林真澄, 保地 眞一
    第104回日本繁殖生物学会  2011/09
  • 顕微授精、精子凍結乾燥、卵子ガラス化の各工程がウシ微小管形成中心の機能発現に及ぼす影響  [Not invited]
    原弘真, 森田大, 桑山正成, 平林真澄, 保地眞一
    第52回日本哺乳動物卵子学会  2011/05
  • IVFまたはICSIに由来するウシ前核期卵におけるDNAメチル化量とH3ヒストンアセチル化量との関係  [Not invited]
    原弘真, 下田美怜, 保地眞一
    第103回日本繁殖生物学会  2010/09
  • ICSIとIVFに由来するウシ胚盤胞の超急速ガラス化保存:発生速度と発育ステージの影響  [Not invited]
    下田美怜, 原弘真, ABDALLA Hany, 森田大, 桑山正成, 平林真澄, 保地眞一
    第51回日本哺乳動物卵子学会  2010/05
  • ICSIとIVFにより作出した拡張度が異なる7日目ウシ胚盤胞におけるガラス化耐性の比較  [Not invited]
    下田 美怜, 原 弘真, ABDALLA Hany, 保地 眞一
    第112回日本畜産学会  2010/03
  • ROCKの活性阻害はガラス化保存した体外受精由来ウシ胚盤胞の蘇生率を改善する  [Not invited]
    原弘真, 下田美怜, ABDALLA Hany, 保地眞一
    第112回日本畜産学会  2010/03

MISC

  • 原弘真, 永山学, 永山学, 永山学, 須田亙, 柴田宏昭, 柴田宏昭, 大貫貴広, 菱川修司, 國田智, 阿部朋行, 阿部朋行, 魚崎英毅, 魚崎英毅, 新幸二, 本田賢也, 本田賢也, 花園豊, 花園豊  日本実験動物学会総会講演要旨集  65th-  157  2018/04  [Not refereed][Not invited]
  • 阿部朋行, 阿部朋行, 柴田宏昭, 原弘真, 魚崎英毅, 魚崎英毅, 大貫貴広, 竹内絢香, 原明日香, SARENTONGLAGA Borjigin, 長尾慶和, 花園豊, 花園豊, 花園豊  日本異種移植研究会プログラム・抄録集  20th-  41  2018  [Not refereed][Not invited]
  • Suvd Byambaa, Hideki Uosaki, Hiromasa Hara, Tomoyuki Abe, Yasumitsu Nagao, Osamu Nureki, Tsukasa Ohmori, Yutaka Hanazono  MOLECULAR THERAPY  25-  (5)  93  -93  2017/05  [Not refereed][Not invited]
  • 阿部朋行, 阿部朋行, 柴田宏昭, 魚崎英毅, 原弘真, 大貫貴広, スブド ビャンバー, ナーウィン ジャントラー, ボラジギン サラントラガ, 福森理加, 長尾慶和, 花園豊, 花園豊, 花園豊  日本実験動物学会総会講演要旨集  64th-  270  2017/04  [Not refereed][Not invited]
  • 原弘真, 柴田宏昭, 中野和明, 阿部朋行, 阿部朋行, 魚崎英毅, 大貫貴広, スブド ビャンバー, ナーウィン ジャントラー, 菱川修司, 國田智, 長嶋比呂志, 濡木理, 花園豊, 花園豊  日本実験動物学会総会講演要旨集  64th-  171  2017/04  [Not refereed][Not invited]
  • 阿部朋行, 阿部朋行, 柴田宏昭, 魚崎英毅, 原弘真, 大貫貴広, SARENTONGLAGA Borjigin, 福森理加, 長尾慶和, 花園豊, 花園豊, 花園豊  日本異種移植研究会プログラム・抄録集  19th-  31  2017  [Not refereed][Not invited]
  • 後藤 哲平, 山口 智之, 佐藤 秀征, 原 弘真, 小林 俊寛, 中内 啓光, 平林 真澄  The Journal of Reproduction and Development  62-  (Suppl.)  j79  -j79  2016/09  [Not refereed][Not invited]
     
    <p>【目的】我々は膵臓欠損(<i>Pdx1 </i>KO)マウス体内にラットiPS細胞由来の膵臓を作製することに成功したが,KOマウス体内で出来たラット膵臓はマウスサイズだったので,移植によりその機能的正常性を検証するだけの膵島を確保できなかった。異種動物に移植臓器を再生させる研究を推進するため,本研究ではTALENによってラットの<i>Pdx1</i>遺伝子をKOし,胚盤胞補完によってマウスiPS細胞に由来する膵臓の再生を試みた。さらに,糖尿病モデルマウスの腎皮膜下に移植したマウスiPS細胞由来膵島の血糖値制御能を検討した。【方法】<i>Pdx1</i>遺伝子上に設計したTALEN Right鎖・Left鎖のRNA,およびExo1 mRNAをCrlj:WIラット由来前核期卵に顕微注入し,<i>Pdx1</i>変異個体を作製した。<i>Pdx1</i>ヘテロ変異個体の交配によって得られた胚盤胞にGFPマウス由来iPS細胞を顕微注入することで,ラット-マウス異種キメラを作製した。キメラ個体の膵臓をコラゲナーゼ処理し,回収した膵島をストレプトゾトシンにより糖尿病誘発したマウスの腎皮膜下に移植し,血糖値の動態を追跡した。【結果】TALENにより7匹の<i>Pdx1</i>遺伝子ヘテロKOラットを得ることに成功した。ヘテロKO個体同士の交配により作製した219個の胚盤胞へマウスiPS細胞を顕微注入し,63匹の生存キメラを得た。<i>Pdx1</i>遺伝子の解析によりこのうち6匹が<i>Pdx1</i>ホモKOラットで,その体内でマウスiPS細胞由来の膵臓が再生されていたことがわかった。糖尿病モデルマウスへ移植したマウスiPS細胞由来膵島は,野生型マウスと同程度にまで血糖値を下げる効果を1年以上にわたって持続した(移植後5日間を除き,免疫抑制剤投与は必要なかった)。以上,胚盤胞補完によって<i>Pdx1 </i>KOラット体内で作製したマウスiPS細胞由来膵島は正常なインシュリン分泌能を有すると証明され,ブタなどの異種動物体内で作製したヒトiPS細胞由来膵島が1型糖尿病の移植治療に使用される可能性を現実的なものにした。</p>
  • Shinichi Hochi, Hany Abdalla, Hiromasa Hara, Masumi Hirabayashi  JOURNAL OF REPRODUCTION AND DEVELOPMENT  57-  (5)  557  -563  2011/10  [Refereed][Not invited]
     
    Freeze-drying (lyophilization) has been proposed as an alternative method for sperm preservation to overcome the disadvantages of the current cryopreservation method such as the high maintenance cost of frozen stocks, the problems associated with transportation of frozen materials and the potential risk of total loss of the frozen stock. Since freeze-dried spermatozoa after rehydration lose their motility, which is an essential requirement to complete physiological fertilization, a relatively difficult microinsemination technique must be applied to rehydrated spermatozoa. Theoretically, it has been supposed that freeze-dried spermatozoa could maintain their functions and abilities to interact with the oocyte cytoplasm after prolonged storage at refrigerator temperature. However, sufficient yield of transferable blastocysts and production of live offspring derived from freeze-dried sperm samples are still subjects to be challenged and overcome in large domestic species.

Awards & Honors

  • 2017/09 メタジェン メタジェン・腸内デザイン賞
     腸内細菌叢ヒト化ブタを用いた腸内環境制御に向けた基盤研究 
    受賞者: 原弘真

Research Grants & Projects

  • アイソレーターを使用しないSCIDブタ長期管理技術の開発
    日本学術振興会:科学研究費補助金 若手研究
    Date (from‐to) : 2019/04 -2021/03 
    Author : 原弘真
  • 腸内細菌叢ヒト化ブタを用いた腸内環境制御に向けた基盤研究
    リバネス:リバネス研究費「メタジェン・腸内デザイン賞」
    Date (from‐to) : 2018/04 -2019/03 
    Author : 原弘真
  • ウシにおける凍結乾燥精子ならびにガラス化卵子由来の胚盤 胞作製システムの構築
    日本学術振興会:科学研究費補助金 特別研究員奨励費
    Date (from‐to) : 2013/04 -2015/03 
    Author : 原弘真
  • ラット多能性幹細胞を利用した臓器欠損モデルの作製と胚盤胞補完法による臓器再生
    日本学術振興会:科学研究費補助金 基盤研究(B)
    Date (from‐to) : 2013/04 -2015/03 
    Author : 平林真澄


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