Researchers Database

hanazono yutaka

    DivisionofRegenerativeMedicine,CenterformolecularMedicine Professor
Last Updated :2021/11/23

Researcher Information


J-Global ID

Research Interests

  • stem cell   regenerative medicine   large animals   swine   iPS cell   hematopoietic stem cell   primate model   sheep   ES cell   hematopoietic stem cell transplantation   

Research Areas

  • Life sciences / Hematology and oncology
  • Life sciences / Laboratory animal science

Academic & Professional Experience

  • 2012  Jichi Medical UniversitySchool of Medicine教授

Published Papers

  • Hitomi Matsunari, Masahito Watanabe, Kazuaki Nakano, Shin Enosawa, Kazuhiro Umeyama, Ayuko Uchikura, Sayaka Yashima, Toru Fukuda, Nikolai Klymiuk, Mayuko Kurome, Barbara Kessler, Annegret Wuensch, Valeri Zakhartchenko, Eckhard Wolf, Yutaka Hanazono, Masaki Nagaya, Akihiro Umezawa, Hiromitsu Nakauchi, Hiroshi Nagashima
    Proceedings of the National Academy of Sciences of the United States of America 115 (4) 708 - 713 1091-6490 2018/01 [Refereed][Not invited]
    Genetically engineered pigs play an indispensable role in the study of rare monogenic diseases. Pigs harboring a gene responsible for a specific disease can be efficiently generated via somatic cell cloning. The generation of somatic cell-cloned pigs from male cells with mutation(s) in an X chromosomal gene is a reliable and straightforward method for reproducing X-linked genetic diseases (XLGDs) in pigs. However, the severe symptoms of XLGDs are often accompanied by impaired growth and reproductive disorders, which hinder the reproduction of these valuable model animals. Here, we generated unique chimeric boars composed of mutant cells harboring a lethal XLGD and normal cells. The chimeric boars exhibited the cured phenotype with fertility while carrying and transmitting the genotype of the XLGD. This unique reproduction system permits routine production of XLGD model pigs through the male-based breeding, thereby opening an avenue for translational research using disease model pigs.
  • Eiji Kobayashi, Yutaka Hanazono, Satoshi Kunita
    Experimental Animals 67 (1) 7 - 13 1341-1357 2018 [Refereed][Not invited]
    Center for Development of Advanced Medical Technology (CDAMTec) in Jichi Medical University was established in 2009. It is the first educational research facility specialized for medical research and training using swine in Japan. Preclinical studies on large animals are essential prior to clinical trials to develop regenerative medical products and medical equipment. We have continued comprehensively considering using miniature swine for experiments to develop advanced medical technologies and train physicians with advanced clinical abilities, while paying attention to animal welfare. The center plays a pioneering role in this field by accumulating know-how such as (1) Construction and effective utilization of research facilities, (2) Procurement of quality animal resources, (3) Education and training of technical staff, (4) Establishment of support system for physicians and researchers. We now open up widely these expertise and foundation for medical research and training not only within our university but also outside the university, so as to move faster to practical use of advanced medical technology and contribute to human health and welfare.
  • 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.
  • Naoki Osada, Jiro Kikuchi, Takashi Umehara, Shin Sato, Masashi Urabe, Tomoyuki Abe, Nakanobu Hayashi, Masahiko Sugitani, Yutaka Hanazono, Yusuke Furukawa
    Oncotarget 9 (5) 6450 - 6462 1949-2553 2018 [Refereed][Not invited]
    Human induced pluripotent stem cells (hiPSCs) are creating great expectations for regenerative medicine. However, safety strategies must be put in place to guard against teratoma formation after transplantation of hiPSC-derived cells into patients. Recent studies indicate that epigenetic regulators act at the initial step of tumorigenesis. Using gain-of-function and loss-of-function approaches, we show here that the expression and function of lysine-specific demethylase 1 (LSD1) are tightly regulated in hiPSCs, and their deregulation underlies the development of teratomas. Consistent with these results, we demonstrate that an LSD1 inhibitor, S2157, prevented teratoma formation from hiPSCs transplanted into immunodeficient mice. This novel action of LSD1 and the effects of its inhibition potentially allow for the development of new clinical applications and therapeutic strategies using hiPSCs.
  • Tomoyuki Abe, Yoshikazu Matsuoka, Yoshikazu Nagao, Yoshiaki Sonoda, Yutaka Hanazono
    INTERNATIONAL JOURNAL OF HEMATOLOGY 106 (5) 631 - 637 0925-5710 2017/11 [Refereed][Not invited]
    We and others have reported that human hematopoietic stem cells (HSCs) are also present in the CD34-negative (CD34(-)) fraction of human cord blood (CB). Here, we examined the hematopoietic engraftment potential of 13 or 18 lineage-negative (13Lin(-) or 18Lin(-)) CD34(+/-) cells from human CB in mice and sheep. Both 13Lin(-) and 18Lin(-) CD34(+) cells efficiently engrafted in mice irrespective of transplantation route, be it by tail-vein injection (TVI) or by intra-bone marrow injection (IBMI). These cells also engrafted in sheep after in utero fetal intra-hepatic injection (IHI). In contrast, neither 13Lin(-) nor 18Lin(-) CD34(-) cells engrafted in either mice or sheep when transplanted by regular routes (i.e., TVI and fetal IHI, respectively), although both 13Lin(-) and 18Lin(-) CD34(-) cells engrafted in mice when transplanted by IBMI and exhibited multilineage reconstitution ability. Thus, the homing ability of CD34(-) HSCs is significantly more limited than that of CD34(+) HSCs. As for 18Lin(-), CD34(-) HSCs are characterized by low expression of the tetraspanin CD9, which promotes homing, and high expression of the peptidase CD26, which inhibits homing. This unique expression pattern homing-related molecules on CD34(-) HSCs could thus explain in part their reduced ability to home to the BM niche.
  • Tsukasa Ohmori, Yasumitsu Nagao, Hiroaki Mizukami, Asuka Sakata, Shin-ichi Muramatsu, Keiya Ozawa, Shin-ichi Tominaga, Yutaka Hanazono, Satoshi Nishimura, Osamu Nureki, Yoichi Sakata
    SCIENTIFIC REPORTS 7 (1) 4159  2045-2322 2017/06 [Refereed][Not invited]
    Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.
  • ABE Tomoyuki, KONO Shota, OHNUKI Takahiro, HISHIKAWA Shuji, KUNITA Satoshi, HANAZONO Yutaka
    Experimental Animals 公益社団法人 日本実験動物学会 0 (0) 345 - 351 1341-1357 2016/11 [Refereed][Not invited]
    Animal models of thrombocytopenia are indispensable for evaluating the in vivo efficacy of hemostatic agents, cryopreserved platelets, and artificial platelets, but no large animal models are available. In this study, we generated a swine model of acute thrombocytopenia with prolonged bleeding times by administering the chemotherapeutic drug busulfan. First, we tested multiple doses of busulfan (4
  • Yoshikazu Arai, Hisho Fukukawa, Takanori Atozi, Shoma Matsumoto, Yutaka Hanazono, Hiroshi Nagashima, Jun Ohgane
    PLOS ONE 11 (1) e0146498  1932-6203 2016/01 [Refereed][Not invited]
    DNA methylation is an epigenetic modification important for cell fate determination and cell type-specific gene expression. Transcriptional regulatory regions of the mammalian genome contain a large number of tissue/cell type-dependent differentially methylated regions (T-DMRs) with DNA methylation patterns crucial for transcription of the corresponding genes. In general, tissues consist of multiple cell types in various proportions, making it difficult to detect T-DMRs of minor cell types in tissues. The present study attempts to detect T-DMRs of minor cell types in tissues by ultra-deep bisulfite sequencing of cell type-restricted genes and to assume proportions of minor cell types based on DNA methylation patterns of sequenced reads. For this purpose, we focused on transcriptionally active hypomethylated alleles (Hypo-alleles), which can be recognized by the high ratio of unmethylated CpGs in each sequenced read (allele). The pituitary gland contains multiple cell types including five hormone-expressing cell types and stem/progenitor cells, each of which is a minor cell type in the pituitary tissue. By ultra-deep sequencing of more than 100 reads for detection of Hypo-alleles in pituitary cell type-specific genes, we identified T-DMRs specific to hormone-expressing cells and stem/progenitor cells and used them to estimate the proportions of each cell type based on the Hypo-allele ratio in pituitary tissue. Therefore, introduction of the novel Hypo-allele concept enabled us to detect T-DMRs of minor cell types with estimation of their proportions in the tissue by ultra-deep bisulfite sequencing.
  • Hanazono Y
    Nihon rinsho. Japanese journal of clinical medicine 73 Suppl 5 360 - 364 0047-1852 2015/06 [Refereed][Not invited]
  • Abe T, Hanazono Y, Nagao Y
    Experimental animals / Japanese Association for Laboratory Animal Science 63 (4) 475 - 481 1341-1357 2014/07 [Refereed][Not invited]
  • Yoshihisa Mizukami, Tomoyuki Abe, Hiroaki Shibata, Yukitoshi Makimura, Shuh-hei Fujishiro, Kimihide Yanase, Shuji Hishikawa, Eiji Kobayashi, Yutaka Hanazono
    PLOS ONE 9 (6) e98319  1932-6203 2014/06 [Refereed][Not invited]
    Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (n = 4) and ovary (n = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (n = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (n = 8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.
  • Yoshikazu Arai, Jun Ohgane, Shuh-Hei Fujishiro, Kazuaki Nakano, Hitomi Matsunari, Masahito Watanabe, Kazuhiro Umeyama, Dai Azuma, Naomi Uchida, Nozomu Sakamoto, Tomohiro Makino, Shintaro Yagi, Kunio Shiota, Yutaka Hanazono, Hiroshi Nagashima
    Genesis 51 (11) 763 - 776 1526-954X 2013/11 [Refereed][Not invited]
    Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor-intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)-specific hypomethylated loci (EShypo-T-DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso-4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso-4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso-622-14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum-free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763-776. © 2013 Wiley Periodicals, Inc.
  • Masahito Watanabe, Kazuaki Nakano, Hitomi Matsunari, Taisuke Matsuda, Miki Maehara, Takahiro Kanai, Mirina Kobayashi, Yukina Matsumura, Rieko Sakai, Momoko Kuramoto, Gota Hayashida, Yoshinori Asano, Shuko Takayanagi, Yoshikazu Arai, Kazuhiro Umeyama, Masaki Nagaya, Yutaka Hanazono, Hiroshi Nagashima
    PLOS ONE 8 (10) e76478  1932-6203 2013/10 [Refereed][Not invited]
    Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration.
  • Kazuaki Nakano, Masahito Watanabe, Hitomi Matsunari, Taisuke Matsuda, Kasumi Honda, Miki Maehara, Takahiro Kanai, Gota Hayashida, Mirina Kobayashi, Momoko Kuramoto, Yoshikazu Arai, Kazuhiro Umeyama, Shuh-hei Fujishiro, Yoshihisa Mizukami, Masaki Nagaya, Yutaka Hanazono, Hiroshi Nagashima
    PLOS ONE 8 (4) e61900  1932-6203 2013/04 [Refereed][Not invited]
    Background: The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naive type characteristics, it is an indispensable challenge to develop naive type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. Methodology/Significant Principal Findings: In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4-8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4-8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively. Conclusion/Significance: Our findings indicate that the aggregation method using parthenogenetic morulae or 4-8 cellstage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.
  • Hanazono Y
    [Rinsho ketsueki] The Japanese journal of clinical hematology 4 54 (4) 329 - 335 0485-1439 2013/04 [Refereed][Not invited]
  • Shuh-hei Fujishiro, Kazuaki Nakano, Yoshihisa Mizukami, Takuya Azami, Yoshikazu Arai, Hitomi Matsunari, Rikiya Ishino, Takashi Nishimura, Masahito Watanabe, Tomoyuki Abe, Yutaka Furukawa, Kazuhiro Umeyama, Shinya Yamanaka, Masatsugu Ema, Hiroshi Nagashima, Yutaka Hanazono
    STEM CELLS AND DEVELOPMENT 22 (3) 473 - 482 1547-3287 2013/02 [Refereed][Not invited]
    In pluripotent stem cells (PSCs), there are 2 types: naive and primed. Only the naive type has the capacity for producing chimeric offspring. Mouse PSCs are naive, but human PSCs are in the primed state. Previously reported porcine PSCs appear in the primed state. In this study, putative naive porcine-induced pluripotent stem cells (iPSCs) were generated. Porcine embryonic fibroblasts were transduced with retroviral vectors expressing Yamanaka's 4 genes. Emergent colonies were propagated in the presence of porcine leukemia inhibitory factor (pLIF) and forskolin. The cells expressed pluripotency markers and formed embryoid bodies, which gave rise to cell types from all 3 embryonic germ layers. The naive state of the cells was demonstrated by pLIF dependency, 2 active X chromosomes (when female), absent MHC class I expression, and characteristic gene expression profiles. The porcine iPSCs contributed to the in vitro embryonic development (11/24, 45.8%) as assessed by fluorescent markers. They also contributed to the in utero fetal development (11/71, 15.5% at day 23; 1/13, 7.7% at day 65). This is the first demonstration of macroscopic fluorescent chimeras derived from naive-like porcine PSCs, although adult chimeras remain to be produced.
  • Miki Maehara, Hitomi Matsunari, Kasumi Honda, Kazuaki Nakano, Yasuhiro Takeuchi, Takahiro Kanai, Taisuke Matsuda, Yukina Matsumura, Yui Hagiwara, Norihisa Sasayama, Akio Shirasu, Masashi Takahashi, Masahito Watanabe, Kazuhiro Umeyama, Yutaka Hanazono, Hiroshi Nagashima
    BIOLOGY OF REPRODUCTION 87 (6) 133  0006-3363 2012/12 [Refereed][Not invited]
    In vitro matured (IVM) oocytes have been used to create genetically modified pigs for various biomedical purposes. However, porcine embryos derived from IVM oocytes are very cryosensitive. Developing improved cryopreservation methods would facilitate the production of genetically modified pigs and also accelerate the conservation of genetic resources. We recently developed a novel hollow fiber vitrification (HFV) method; the present study was initiated to determine whether this new method permits the cryopreservation of IVM oocyte-derived porcine embryos. Embryos were created from the in vitro fertilization of IVM oocytes with frozen-thawed sperm derived from a transgenic pig carrying a humanized Kusabira-Orange (huKO) gene. Morula-stage embryos were assigned to vitrification and nonvitrification groups to compare their in vitro and in vivo developmental abilities. Vitrified morulae developed to the blastocyst stage at a rate similar to that of nonvitrified embryos (66/85, 77.6% vs. 67/84, 79.8%). Eighty-eight blastocysts that developed from vitrified morulae were transferred into the uteri of three recipient gilts. All three became pregnant and produced a total of 17 piglets (19.3%). This piglet production was slightly lower, albeit not significantly, than that of the nonvitrification group (27/88, 30.7%). Approximately half of the piglets in the vitrification (10/17, 58.8%) and nonvitrification (15/27, 55.6%) groups were transgenic. There was no significant difference in the growth rates among the piglets in the two groups. These results indicate that the HFV method is an extremely effective method for preserving cryosensitive embryos such as porcine in vitro maturation/fertilization-derived morulae.
  • Tomoyuki Abe, Shigeo Masuda, Yujiro Tanaka, Suguru Nitta, Yoshihiro Kitano, Satoshi Hayashi, Yutaka Hanazono, Yoshikazu Nagao
    EXPERIMENTAL HEMATOLOGY 40 (6) 436 - 444 0301-472X 2012/06 [Refereed][Not invited]
    In utero transplantation (IUT) of human hematopoietic stem cells has been conducted in sheep, which are used as large animal models of human hematopoietic reconstitution and models for clinical IUT; however, the levels of engraftment have generally been low. Busulfan (BU), a myeloablative agent, is often administered to patients before hematopoietic stem cells transplantation to improve the engraftment. In this study, hematopoietic activity was evaluated in adult sheep after administering BU at different doses. Next, pregnant ewes were administered BU, and dams as well as their fetuses were evaluated, as BU readily crosses the sheep placenta. Then, the BU dose with the desired outcomes was selected and administered to pregnant ewes at 2 or 6 days before performing JUT using human cord blood CD34(+) cells. The engraftment was evaluated in recipients that underwent JUT in the presence or absence of BU. As a result, hematopoietic activity was safely and transiently suppressed in adult sheep treated with 5 to 7.5 mg/kg BU. BU crossed the sheep placenta, and fetal sheep were indeed conditioned by administering 3 mg/kg BU to pregnant ewes. Engraftment of human CD34(+) cells in fetal recipients was enhanced when IUT was carried out 6 days post-BU. Up to 3.3% engraftment levels (in terms of bone marrow colony-forming units) were achieved with the JUT of 0.72 to 2.4 million CD34(+) cells when BU was used. BU can be administered to pregnant ewes to effectively condition the fetal recipient for IUT with enhanced engraftment of donor cells. (C) 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
  • Masuda S, Yokoo T, Sugimoto N, Doi M, Fujishiro SH, Takeuchi K, Kobayashi E, Hanazono Y
    Cell medicine 3 (1-3) 103 - 112 2012/01 [Refereed][Not invited]
  • Shigeo Masuda, Satoshi Hayashi, Naohide Ageyama, Hiroaki Shibata, Tomoyuki Abe, Yoshikazu Nagao, Yutaka Hanazono
    Transplantation 92 (2) e5-6; author reply e6-7 - 6; author reply e6 0041-1337 2011/07 [Refereed][Not invited]
  • Masuda S, Hanazono Y
    The New England journal of medicine 364 (2) 181; author reply 181 - 2 0028-4793 2011/01 [Refereed][Not invited]
  • Tomoyuki Abe, Shigeo Masuda, Hiroshi Ban, Satoshi Hayashi, Yasuji Ueda, Makoto Inoue, Mamoru Hasegawa, Yoshikazu Nagao, Yutaka Hanazono
    EXPERIMENTAL HEMATOLOGY 39 (1) 47 - 54 0301-472X 2011/01 [Refereed][Not invited]
    Objective The homeobox B4 (HoxB4) gene promotes expansion of hematopoietic stem cells (HSCs) However, frequent development of leukemia in large animals duo to retrovirally transduced HoxB4 gene has been reported To prevent tumorigenesis, we developed a nonintegrating and nonreplicating Sendai virus vector that did not contain the phosphoprotein gene (SeV/Delta P), which enabled clearance of the vector and transgene shortly after transduction We tested the SeV/Delta P vector expressing the HoxB4 gene (SeV/Delta P/HoxB4) for the ex vivo expansion of human cord blood CD34(+) cells (HSCs) using a sheep in utero transplantation assay Materials and Methods Human HSCs were ex vivo expanded by transduction with SeV/Delta P/HoxB4 vector and transplanted into the abdominal cavity of fetal sheep The. engraftment of human HSCs in the lambs was quantitatively evaluated by hematopoietic colony forming unit assays Results After transplantation, the HoxB4-transduced HSCs contributed to longer period (up to 20 months) repopulation in sheep, and human hematopoietic progenitors were detected more frequently in the bone marrow of the HoxB4 group as compared with the control untreated group (p < 0 05) The expansion of human HSCs with the SeV/Delta P/HoxB4 vector was comparable with previously reported retroviral vectors expressing HoxB4 The SeV/Delta P/HoxB4 vector and the transgene were cleared from the recipient sheep and leukemia was not detected at 20 months post transplantation Conclusions The SeV/Delta P vector would be suitable for transient expression of HoxB4 in human CD34(+) cells In addition, the SeV/Delta P vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors (C) 2011 ISFH Society for Hematology and Stem Cells Published by Elsevier Inc
  • Masuda S, Hanazono Y
    The New England journal of medicine 2 364 (2) 181; author reply 181 - 2 0028-4793 2011/01 [Refereed][Not invited]
  • Yutaka Hanazono
    Seikagaku 83 (11) 1060 - 1063 0037-1017 2011 [Refereed][Not invited]
  • Yujiro Tanaka, Shigeo Masuda, Tomoyuki Abe, Satoshi Hayashi, Yoshihiro Kitano, Yoshikazu Nagao, Yutaka Hanazono
    TRANSPLANTATION 90 (4) 462 - 463 0041-1337 2010/08 [Refereed][Not invited]
  • Shigeo Masuda, Keiki Kumano, Takahiro Suzuki, Taisuke Tomita, Takeshi Iwatsubo, Hideaki Natsugari, Arinobu Tojo, Makoto Shibutani, Kunitoshi Mitsumori, Yutaka Hanazono, Seishi Ogawa, Mineo Kurokawa, Shigeru Chiba
    CANCER SCIENCE 100 (12) 2444 - 2450 1347-9032 2009/12 [Refereed][Not invited]
    Constitutive activation of Notch signaling is required for the proliferation of a subgroup of human T-cell acute lymphoblastic leukemias (T-ALL). Previous in vitro studies have demonstrated the therapeutic potential of Notch signaling inhibitors for treating T-ALL. To further examine this possibility, we applied a gamma-secretase inhibitor (GSI) to T-ALL xenograft models. Treatment of established subcutaneous tumors with GSI resulted in partial or complete regression of tumors arising from four T-ALL cell lines that were also sensitive to GSI in vitro. To elucidate the mechanism of action, we transduced DND-41 cells with the active form of Notch1 (aN1), which conferred resistance to in vitro GSI treatment. Nevertheless, in vivo treatment with GSI induced a partial but significant regression of subcutaneous tumors that developed from aN1-transduced DND-41 cells, whereas it induced complete regression of tumors that developed from mock-transduced DND-41 cells. These findings indicate that the remarkable efficacy of GSI might be attributable to dual mechanisms, directly via apoptosis of DND-41 cells through the inhibition of cell-autonomous Notch signaling, and indirectly via disturbance of tumor angiogenesis through the inhibition of non-cell-autonomous Notch signaling. (Cancer Sci 2009; 100: 2444-2450).
  • Shigeo Masuda, Naohide Ageyama, Hiroaki Shibata, Yoko Obara, Tamako Ikeda, Kengo Takeuchi, Yasuji Ueda, Keiya Ozawa, Yutaka Hanazono
    Experimental hematology 37 (10) 1250 - 1257 0301-472X 2009/10 [Refereed][Not invited]
    OBJECTIVE: Hematopoietic stem cells (HSCs) reside in the osteoblastic niche, which consists of osteoblasts. Mesenchymal stromal cells (MSCs) have an ability to differentiate into osteoblasts. Here, using nonhuman primates, we investigated the effects of cotransplantation with MSCs on the engraftment of HSCs after autologous intra-bone marrow transplantation. MATERIALS AND METHODS: From three cynomolgus monkeys, CD34-positive cells (as HSCs) and MSCs were obtained. The former were divided into two equal aliquots and each aliquot was genetically marked with a distinctive retroviral vector to track the in vivo fate. Each HSC aliquot with or without MSCs was autologously injected into the bone marrow (BM) cavity of right or left side, enabling the comparison of in vivo fates of the two HSC grafts in the same body. RESULTS: In the three monkeys, CD34(+) cells transplanted with MSCs engrafted 4.4, 6.0, and 1.6 times more efficiently than CD34(+) cells alone, as assessed by BM colony polymerase chain reaction. In addition, virtually all marked cells detected in the peripheral blood were derived from the cotransplantation aliquots. Notably, colony-forming units derived from the cotransplantation aliquots were frequently detected in BM distant sites from the injection site, implying that cotransplantation with MSCs also restored the ability of gene-marked HSCs to migrate and achieve homing in the distant BM. CONCLUSION: Cotransplantation with MSCs would improve the efficacy of transplantation of gene-modified HSCs in primates, with enhanced engraftment in BM as well as increased chimerism in peripheral blood through migration and homing.
  • Yoshikazu Nagao, Tomoyuki Abe, Hideaki Hasegawa, Yujiro Tanaka, Kyoko Sasaki, Yoshihiro Kitano, Satoshi Hayashi, Yutaka Hanazono
    CLONING AND STEM CELLS 11 (2) 281 - 285 1536-2302 2009/06 [Refereed][Not invited]
    In the present study, we investigated the suitability of two methods for the transplantation of cells into ovine fetuses. The first method was an ultrasound-guided cell injection via the uterine wall. The second involved hysterotomic cell injection with an incision in the uterine wall exposing the amnion. Monkey embryonic stem (ES) cell-derived hematopoietic cells were used as donor cells. After transplantation, the abortion rate associated with the hysterotomic injection method was significantly higher than that of the ultrasound-guided injection method (8/13 versus 4/24; P < 0.01). The fetuses were delivered to examine the engraftment of transplanted monkey hematopoietic cells. Monkey cells were detected in one of the five animals (20%) in the hysterotomic injection group, and 14 of 20 animals (70%, P < 0.05) in the ultrasound-guided injection group. Therefore, the ultrasound-guided method was effectively shown to be minimally invasive for in utero transplantation and can produce a higher rate of engraftment for transplanted cells.
  • Yujiro Tanaka, Tamako Ikeda, Yukiko Kishi, Shigeo Masuda, Hiroaki Shibata, Kengo Takeuchi, Makoto Komura, Tadashi Iwanaka, Shin-ichi Muramatsu, Yasushi Kondo, Kazutoshi Takahashi, Shinya Yamanaka, Yutaka Hanazono
    CELL TRANSPLANTATION 18 (4) 381 - 389 0963-6897 2009 [Refereed][Not invited]
    The ERas gene promotes the proliferation of and formation of teratomas by Mouse embryonic stem (ES) cells. However, its human orthologue is not expressed in human ES cells. This implies that the behavior of transplanted mouse ES cells would not accurately reflect the behavior of transplanted human ES cells and that the use of nonhuman primate models might be more appropriate to demonstrate the safety of human ES cell-based therapies. However, the expression of the ERas gene has not been examined in nonhuman primate ES cells. In this study, we cloned the cynomolgus homologue and showed that the ERas gene is expressed in cynomolgus ES cells. Notably, it is also expressed in cynomolgus ES cell-derived differentiated progeny as well as cynomolgus adult tissues. The ERas protein is detectable in various cynomolgus tissues its assessed by immunohistochemisty. Cynomolgus ES cell-derived teratoma cells, which also expressed the ERas gene at higher levels than the undifferentiated cynomolgus ES cells, did not develop tumors in NOD/Shi-scid, IL-2R gamma(null) (NOG) mice. Even when the ERas gene was overexpressed in cynomolgus stromal cells, only the plating efficiency was improved and the proliferation was not promoted. Thus, it is unlikely that ERas contributes to the tumorigenicity of cynomolgus cells. Therefore, cynomolgus ES cells are more similar to human than mouse ES cells despite that ERas is expressed in cynomolgus and mouse ES cells but not in human ES cells.
  • Yukiko Kishi, Makoto Inoue, Yujiro Tanaka, Hiroaki Shibata, Shigeo Masuda, Tamako Ikeda, Mamoru Hasegawa, Yutaka Hanazono
    CLONING AND STEM CELLS 10 (3) 307 - 312 1536-2302 2008/09 [Refereed][Not invited]
    Sendai virus (SeV) vectors can introduce foreign genes efficiently and stably into primate embryonic stern (ES) cells. For the application of these cells, the control of transgene expression is important. Cynomolgus ES cells transduced with a SeV vector expressing the green fluorescent protein (GFP) gene were propagated in Knockout serum replacement (KSR)-supplemented medium, used widely for the serum-free culture of ES cells, and growth and transgene expression were evaluated. The SeV vector-mediated GFP expression was suppressed in the KSR-supplemented medium, although it was stable in regular fetal bovine serum (FBS)-supplemented medium. Propagation in the KSR-supplemented medium eventually resulted in a complete suppression of GFP expression and eradication of the SeV genome. The inhibitory effect of KSR on the transduction was attributable to the positive selection of untransduced ES cells in addition to the removal of the SeV vector from transduced cells. KSR also reduced the efficiency of the transduction. SeV vector-mediated transgene expression in ES cells was suppressed in the KSR-supplemented medium. Although the suppression is limited in specified cells such as ES cells, these findings will help elucidate how to control transgene expression.
  • Yujiro Tanaka, Shinichiro Nakamura, Hiroaki Shibata, Yukiko Kishi, Tamako Ikeda, Shigeo Masuda, Kyoko Sasaki, Tomoyuki Abe, Satoshi Hayashi, Yoshihiro Kitano, Yoshikazu Nagao, Yutaka Hanazono
    STEM CELLS AND DEVELOPMENT 17 (2) 367 - 381 1547-3287 2008/04 [Refereed][Not invited]
    Because embryonic stem (ES) cells are able to proliferate indefinitely and differentiate into any type of cell, they have the potential for providing an inexhaustible supply of transplantable cells or tissues. However, methods for the in vitro differentiation of human ES cells are still quite limited. One possible strategy would be to generate differentiated cells in vivo. In view of future clinical application, we investigated the possibility of using xenogeneic large animals for this purpose. We transplanted nonhuman primate cynomolgus ES cells into fetal sheep at 43-67 gestational days ( full term 147 days, n = 15). After birth, cynomolgus tissues, which were mature teratomas, had been engrafted in sheep when more than 1 X 10(6) ES cells were transplanted at < 50 gestational days. Despite the sustained engraftment, both cellular and humoral immune responses against the ES cells were detected, and additional transplantation was not successful in the animals. At 2 weeks post-transplantation, the ES cell progeny proliferated when transplanted at 48 ( < 50) gestational days, whereas they were cleared away when transplanted at 60 ( < 50) gestational days. These results support the rapid development of the xenogeneic immunological barrier in fetal sheep after 50 gestational days. Notably, a large number of Foxp3(+) regulatory T cells were present around the ES cell progeny, but macrophages were absent when the transplant was conducted at < 50 gestational days, implying that regulatory T cells and premature innate immunity might have contributed to the sustained engraftment. In conclusion, long-term macroscopic engraftment of primate ES cells in sheep is feasible despite the xenogeneic immunological barrier.
  • Hanazono Y
    [Rinsho ketsueki] The Japanese journal of clinical hematology 4 49 240 - 246 0485-1439 2008/04 [Refereed][Not invited]
  • Yukiko Kishi, Yujiro Tanaka, Hiroaki Shibata, Shinichiro Nakamura, Koichi Takeuchi, Shigeo Masuda, Tamako Ikeda, Shin-ichi Muramatsu, Yutaka Hanazono
    CELL TRANSPLANTATION 17 (9) 1095 - 1102 0963-6897 2008 [Refereed][Not invited]
    Embryonic stem (ES) cells have the ability to generate teratomas when transplanted into immunodeficient mice, but conditions affecting the generation remain to be elucidated. Nonhuman primate cynomolgus ES cells were transplanted into immunodeficient mice under different conditions; the number of transplanted cells, physical state (clumps or single dissociated cells), transplant site, differentiation state, and immunological state of recipient mice were all varied. The tumorigenicity was then evaluated. When cynomolgus ES cells were transplanted as clumps into the lower limb muscle in either nonobese diabetic/severe combined immunodeficiency (NOD/SCID) or NOD/SCID/gamma c(null) (NOG) mice, teratomas developed in all the animals transplanted with 1 x 10(5) or more cells, but were not observed in any mouse transplanted with 1 x 10(3) cells. However, when the cells were transplanted as dissociated cells, the number of cells necessary for teratornas to form in all mice increased to 5 x 10(5). When the clump cells were injected. subcutaneously (instead of intramuscularly), the number also increased to 5 x 10(5). When cynomolgus ES cell-derived progenitor cells (1 x 10(6)), which included residual pluripotent cells, were transplanted into the lower limb muscle of NOG or NOD/SCID mice, the incidence of teratomas differed between the strains; teratomas developed in five of five NOG mice but in only two of five NOD/SCID mice. The incidence of teratomas varied substantially depending on the transplanted cells and recipient mice. Thus, considerable care must be taken as to tumorigenicity.
  • Hiroaki Shibata, Naohide Ageyama, Yujiro Tanaka, Yukiko Kishi, Kyoko Sasaki, Shinichiro Nakamura, Shin-ichi Muramatsu, Satoshi Hayashi, Yoshihiro Kitano, Keiji Terao, Yutaka Hanazono
    Stem cells (Dayton, Ohio) 24 (6) 1450 - 7 1066-5099 2006/06 [Refereed][Not invited]
    Cynomolgus monkey embryonic stem cell (cyESC)-derived in vivo hematopoiesis was examined in an allogeneic transplantation model. cyESCs were induced to differentiate into the putative hematopoietic precursors in vitro, and the cells were transplanted into the fetal cynomolgus liver at approximately the end of the first trimester (n = 3). Although cyESC-derived hematopoietic colony-forming cells were detected in the newborns (4.1%-4.7%), a teratoma developed in all newborns. The risk of tumor formation was high in this allogeneic transplantation model, given that tumors were hardly observed in immunodeficient mice or fetal sheep that had been xeno-transplanted with the same cyESC derivatives. It turned out that the cyESC-derived donor cells included a residual undifferentiated fraction positive for stage-specific embryonic antigen (SSEA)-4 (38.2% +/- 10.3%) despite the rigorous differentiation culture. When an SSEA-4-negative fraction was transplanted (n = 6), the teratoma was no longer observed, whereas the cyESC-derived hematopoietic engraftment was unperturbed (2.3%-5.0%). SSEA-4 is therefore a clinically relevant pluripotency marker of primate embryonic stem cells (ESCs). Purging pluripotent cells with this surface marker would be a promising method of producing clinical progenitor cell preparations using human ESCs.
  • Naohide Ageyama, Yutaka Hanazono, Hiroaki Shibata, Fumiko Ono, Takeyuki Nagashima, Yasuji Ueda, Yasuhiro Yoshikawa, Mamoru Hasegawa, Keiya Ozawa, Keiji Terao
    The Journal of veterinary medical science 68 (5) 507 - 10 0916-7250 2006/05 [Refereed][Not invited]
    Genes and proteins of human origin are often administered to monkeys for research purposes, however, it can be difficult to obtain sufficient levels of the products in vivo due to immunological clearance. In this study, we showed that human erythropoietin (hEPO) induces generation of anti-hEPO antibody in cynomolgus macaques (n=2), although 92% of amino acid residues are common between the human and macaque EPO. The administered hEPO was thus eliminated from the animals. On the other hand, when an immunosuppressant, cyclosporin A (CyA), was administered (6 mg/kg) intramuscularly every other day in combination with hEPO (n=2), no anti-hEPO antibody was generated and high serum levels of hEPO were obtained during administration of hEPO, resulting in an increase in serum hemoglobin levels. No adverse effects associated with CyA were observed. Thus, CyA treatment is useful for prevention of immune responses associated with the administration of human proteins in monkeys.
  • Asano T, Shibata H, Hanazono Y
    Methods in molecular biology (Clifton, N.J.) 329 295 - 303 1064-3745 2006 [Refereed][Not invited]
  • Asano T, Sasaki K, Kitano Y, Terao K, Hanazono Y
    Methods in molecular biology (Clifton, N.J.) 329 459 - 467 1064-3745 2006 [Refereed][Not invited]
  • Naohide Ageyama, Yutaka Hanazono, Hiroaki Shibata, Fumiko Ono, Hiromi Ogawa, Takeyuki Nagashima, Yasuji Ueda, Yasuhiro Yoshikawa, Mamoru Hasegawa, Keiya Ozawa, Keiji Terao
    Experimental animals 54 (5) 421 - 8 1341-1357 2005/10 [Refereed][Not invited]
    Hematopoietic stem cells in bone marrow can be mobilized into peripheral blood by cytokine administration. Cytokine-mobilized peripheral blood stem cells are of great use in clinical applications. We previously established a modified procedure for the collection of cytokine-mobilized peripheral blood cells from rhesus monkeys (Macaca mulata) using a commercially available apparatus originally developed for human subjects. In this study, we examined the efficacy and safety of this method with even smaller macaques, cynomolgus monkeys (Macaca fascicularis), which are equivalent to human newborns in body weight (mean = 3.3 kg). Using the manufacturer's unmodified protocol (n=6), one monkey died of cardiac failure and three developed severe anemia. In contrast, using our modified procedure (n=6), no such complication was observed in any animal. In addition, the harvested nuclear cell, mononuclear cell and CD34(+) cell counts were significantly higher with the modified method. The modified method should allow safe and efficient collection of cytokine-mobilized peripheral blood cells from non-human primates as small as human newborns in a non-invasive manner.
  • Toru Yoshioka, Naohide Ageyama, Hiroaki Shibata, Takanori Yasu, Yoshio Misawa, Koichi Takeuchi, Keiji Matsui, Keiji Yamamoto, Keiji Terao, Kazuyuki Shimada, Uichi Ikeda, Keiya Ozawa, Yutaka Hanazono
    Stem cells (Dayton, Ohio) 23 (3) 355 - 64 1066-5099 2005/03 [Refereed][Not invited]
    Rodent and human clinical studies have shown that transplantation of bone marrow stem cells to the ischemic myocardium results in improved cardiac function. In this study, cynomolgus monkey acute myocardial infarction was generated by ligating the left anterior descending artery, and autologous CD34(+) cells were transplanted to the peri-ischemic zone. To track the in vivo fate of transplanted cells, CD34(+) cells were genetically marked with green fluorescent protein (GFP) using a lentivirus vector before transplantation (marking efficiency, 41% on average). The group receiving cells (n = 4) demonstrated improved regional blood flow and cardiac function compared with the saline-treated group (n =4) at 2 weeks after transplant. However, very few transplanted cell-derived, GFP-positive cells were found incorporated into the vascular structure, and GFP-positive cardiomyocytes were not detected in the repaired tissue. On the other hand, cultured CD34(+) cells were found to secrete vascular endothelial growth factor (VEGF), and the in vivo regional VEGF levels showed a significant increase after the transplantation. These results suggest that the improvement is not the result of generation of transplanted cell-derived endothelial cells or cardiomyocytes; and raise the possibility that angiogenic cytokines secreted from transplanted cells potentiate angiogenic activity of endogenous cells.
  • K Sasaki, Y Nagao, Y Kitano, H Hasegawa, H Shibata, M Takatoku, S Hayashi, K Ozawa, Y Hanazono
    TRANSPLANTATION 79 (1) 32 - 37 0041-1337 2005/01 [Refereed][Not invited]
    Background. Although directed differentiation of human embryonic stem (ES) cells would enable a ready supply of cells and tissues required for transplantation therapy, the methodology is limited. We have developed a novel method for hematopoietic development from primate ES cells. We first cultured cynomolgus monkey ES cells in vitro and transplanted the cells in vivo into fetal sheep liver, generating sheep with cynomolgus hematopoiesis. Methods. Cynomolgus ES cells were induced to mesodermal cells on murine stromal OP9 cells with multiple cytokines for 6 days. The cells (average 4.8 x 10(7) cells) were transplanted into fetal sheep in the liver (n = 4) after the first trimester (day 55-73, full term 147 days). The animals were delivered at full term, and two of them were intraperitoneally administered with human stem-cell factor (SCF). Results. Cynomolgus hematopoietic progenitor cells were detected in bone marrow at a level of 1% to 2% in all four sheep up to 17 months posttransplant. No teratoma was found in the lambs. After SCF administration, the fractions of cynomolgus hematopoiesis increased by several-fold (up to 13%). Cynomolgus cells were also detected in the circulation, albeit at low levels ( < 0.1%). Conclusions. Long-term hematopoietic microchimerism from primate ES cells was observed after in vitro differentiation to mesodermal cells, followed by in vivo introduction into the fetal liver microenvironment. The mechanism of such directed differentiation of ES cells remains to be elucidated, but this procedure should allow further investigation.
  • J Kikuchi, J Mimuro, K Ogata, T Tabata, Y Ueda, A Ishiwata, K Kimura, K Takano, S Madoiwa, H Mizukami, Y Hanazono, A Kume, M Hasegawa, K Ozawa, Y Sakata
    JOURNAL OF GENE MEDICINE 6 (10) 1049 - 1060 1099-498X 2004/10 [Refereed][Not invited]
    Background Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34(+) cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). Methods CD34(+) cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34(+) cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34(+) cells and expression of transgenes were studied. Results We could efficiently transduce CD34(+) cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34(+) cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 106 CD34(+) cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34(+) cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34(+) cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. Conclusions Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients. Copyright (C) 2004 John Wiley Sons, Ltd.
  • T Kanazawa, H Mizukami, H Nishino, T Okada, Y Hanazono, A Kume, K Kitamura, K Ichimura, K Ozawa
    INTERNATIONAL JOURNAL OF ONCOLOGY 25 (3) 729 - 735 1019-6439 2004/09 [Refereed][Not invited]
    Adeno-associated virus (AAV) is a non-pathogenic virus with a single-strand DNA genome. AAV vectors have several unique properties suited for gene therapy applications. However, an obstacle to their application is a low efficiency of transgene expression, mainly due to a limited second-strand synthesis. Previously, we reported that gamma-rays enhanced the transduction efficiency and cytocidal effect of AAV vector harboring the herpes simplex virus-thymidine kinase (AAVtk) and ganciclovir (GCV) system. In the present study, we investigated whether topoisomerase inhibitors (etoposide and camptothecin) enhance the AAV vector-mediated transgene expression and the killing effect by AAVtk/GCV system. The enhancement of transgene expression was observed in a concentration -dependent manner on human laryngeal carcinoma cells (HEp-2 cells) and HeLa cells. Southern analysis confirmed that etoposide enhanced the double-strand synthesis of the AAV vector genome in HEp-2 cells and HeLa cells. The cells were efficiently killed by AAVtk/GCV system, as expected. More importantly, both etoposide and camptothecin augmented the cytocidal effect of the AAVtk/GCV system. These findings suggest that the combination of AAV-mediated suicide gene therapy and treatment with topoisomerase inhibitors may have synergistic therapeutic effects in the treatment of cancers.
  • Kyoji Ueda, Yutaka Hanazono, Hiroaki Shibata, Naohide Ageyama, Yasuji Ueda, Satoko Ogata, Toshiaki Tabata, Takeyuki Nagashima, Masaaki Takatoku, Akihiko Kume, Susumu Ikehara, Masafumi Taniwaki, Keiji Terao, Mamoru Hasegawa, Keiya Ozawa
    Molecular therapy : the journal of the American Society of Gene Therapy 10 (3) 469 - 77 1525-0016 2004/09 [Refereed][Not invited]
    The successful engraftment of genetically modified hematopoietic stem cells (HSCs) without toxic conditioning is a desired goal for HSC gene therapy. To this end, we have examined the combination of intrabone marrow transplantation (iBMT) and in vivo expansion by a selective amplifier gene (SAG) in a nonhuman primate model. The SAG is a chimeric gene consisting of the erythropoietin (EPO) receptor gene (as a molecular switch) and c-Mpl gene (as a signal generator). Cynomolgus CD34+ cells were retrovirally transduced with or without SAG and returned into the femur and humerus following irrigation with saline without prior conditioning. After iBMT without SAG, 2-30% of colony-forming cells were gene marked over 1 year. The marking levels in the peripheral blood, however, remained low (<0.1%). These results indicate that transplanted cells can engraft without conditioning after iBMT, but in vivo expansion is limited. On the other hand, after iBMT with SAG, the peripheral marking levels increased more than 20-fold (up to 8-9%) in response to EPO even at 1 year posttransplant. The increase was EPO-dependent, multilineage, polyclonal, and repeatable. Our results suggest that the combination of iBMT and SAG allows efficient in vivo gene transduction without marrow conditioning.
  • Takeyuki Nagashima, Yasuji Ueda, Yutaka Hanazono, Akihiro Kume, Hiroaki Shibata, Naohide Ageyama, Keiji Terao, Keiya Ozawa, Mamoru Hasegawa
    The journal of gene medicine 6 (1) 22 - 31 1099-498X 2004/01 [Refereed][Not invited]
    BACKGROUND: In vivo expansion of gene-modified cells would be a promising approach in the field of hematopoietic stem cell gene therapy. To this end, we previously developed a selective amplifier gene (SAG), a chimeric gene encoding the granulocyte colony-stimulating factor (G-CSF) receptor (GCR), as a growth-signal generator and the hormone-binding domain of the steroid receptor as a molecular switch. We have already reported that hematopoietic cells retrovirally transduced with the SAG can be expanded in a steroid-dependent manner in vitro and in vivo in mice and nonhuman primates. In this study, we have developed a new-generation SAG, in which the erythropoietin (EPO) receptor (EPOR) is utilized instead of the steroid receptor as a molecular switch. METHODS: Two EPO-driven SAGs were constructed, EPORGCR and EPORMpl, containing the GCR and c-Mpl as a signal generator, respectively. First, to compare the steroid-driven and EPO-driven SAGs, Ba/F3 cells were transduced with these SAGs. Next, to examine whether GCR or c-Mpl is the more suitable signal generator of the EPO-driven SAG, human cord blood CD34(+) cells were transduced with the two EPO-driven SAGs (EPORMpl and EPORGCR). Finally, we examined the in vivo efficacy of EPORMpl in mice. Irradiated mice were transplanted with EPORMpl-transduced bone marrow cells followed by administration of EPO. RESULTS: The EPO-driven SAGs were shown to induce more rapid and potent proliferation of Ba/F3 cells than the steroid-driven SAGs. The EPORMpl induced more efficient EPO-dependent proliferation of the human cord blood CD34(+) cells than the EPORGCR in terms of total CD34(+) cell, c-Kit(+) cell, and clonogenic progenitor cell (CFU-C) numbers. In the transplanted mice the transduced peripheral blood cells significantly increased in response to EPO. CONCLUSIONS: The new-generation SAGs, especially EPORMpl, are able to efficiently confer an EPO-dependent growth advantage on transduced hematopoietic cells in vitro and in vivo in mice.
  • J Ideno, H Mizukami, K Honda, T Okada, Y Hanazono, A Kume, T Saito, S Ishibashi, K Ozawa
    MOLECULAR THERAPY 8 (6) 895 - 902 1525-0016 2003/12 [Refereed][Not invited]
    Adeno-associated virus (AAV) vector is suitable for gene transfer to the central nervous system. However, the efficacy of gene therapy for neuroendocrine disease is still unknown. In this study, we injected AAV vector encoding arginine-vasopressin (AVP) stereotaxically into the bilateral hypothalamus of Brattleboro rats. Brattleboro rats show a central diabetes insipidus (CDI) phenotype and growth retardation due to a complete deficiency of AVP. Following injection, both urine volume and urine osmolality normalized, and these therapeutic effects persisted for more than 50 weeks. In addition to phenotypic correction, secretion of transgene-derived AVP was enhanced after 24 h water deprivation or hypertonic saline injection, and water diuresis was demonstrated after acute water loading. Also, reduced body weight and low plasma insulin-like growth factor 1 levels of Brattleboro rats were restored after AVP gene transduction, suggesting the importance of AVP in growth. These findings indicate that hypothalamic neurons of Brattleboro rats can produce and release mature AVP following AAV-mediated gene transduction, resulting in long-term phenotypic correction of CDI. Moreover, the fact that transgene-derived AVP was secreted adequately in response to stimuli, even if it was expressed constitutively, suggests advantages of gene therapy for neuroendocrine diseases and offers a basis to investigate AVP function.
  • M Nagata, MA Takahashi, S Muramatsu, Y Ueda, Y Hanazono, K Takeuchi, K Okada, Y Suzuki, Y Kondo, M Suemori, U Ikeda, Nakano, I, E Kobayashi, M Hasegawa, K Ozawa, N Nakatsuji, K Shimada
    JOURNAL OF GENE MEDICINE 5 (11) 921 - 928 1099-498X 2003/11 [Refereed][Not invited]
    Background Embryonic stem (ES) cells continually proliferate and can generate large numbers of differentiated cells. Genetic manipulation of transplantable cells derived from primate ES cells offers considerable potential for development research and regenerative cell therapy. However, protocols for efficient gene transfer into primate ES-cell-derived cells have not yet been established. Methods Spontaneously contracting areas were derived from cynomolgus monkey ES cells. Features of cardiomyocytes in the area were analyzed according to gene expression (RT-PCR), morphology (immunostaining and electron microscopy), and function (intracellular calcium transience). Beating cells were transduced using a simian immunodeficiency virus (STV) vector expressing enhanced green fluorescence protein (EGFP), then transplanted into ischemic rat myocardium. Results Beating cells derived from monkey ES cells displayed gene expression, ultrastructural and functional properties of early-stage cardiomyocytes. Highly efficient (97% cardiac phenotype) and stable transduction of these ES-cell-derived cardiomyocytes was achieved using SIV vector without altering contractile function. In addition, transduced cardiomyocytes survived in the myocardium of a rat myocardial infarction model. Conclusions A lentiviral vector system based on SIV represents a useful vehicle for genetic modification of cardiomyocytes derived from primate ES cells, and can extend the application of primate ES cells to gene therapy. Copyright (C) 2003 John Wiley Sons, Ltd.
  • A Itoh, T Okada, H Mizuguchi, T Hayakawa, H Mizukami, A Kume, M Takatoku, N Komatsu, Y Hanazono, K Ozawa
    JOURNAL OF GENE MEDICINE 5 (11) 929 - 940 1099-498X 2003/11 [Refereed][Not invited]
    Background Although adenoviral vectors primarily derived from the adenovirus serotype 5 (Ad5) are widely used for many gene transfer applications, they cannot efficiently infect hematopoietic cells, since these cells do not express the coxsackie-adenoviral receptor (CAR). Methods We have developed a soluble fusion protein that bridges adenoviral fibers and the c-Kit receptor to alter Ad5 tropism to immature hematopoietic cells. The CAR-SCF fusion protein consists of the extracellular domains of CAR and stem cell factor (SCF). The human megakaryoblastic leukemia cell lines UT-7 and M07e, human chronic myelogenous leukemia cell line K-562, and erythroleukemia cell line TF-1 were used to assess CAR-SCF-assisted Ad5-mediated gene transfer. Hematopoietic cell lines were infected with an Ad5 vector (Ad5-eGFP) or a fiber-mutant Ad5/F35 (Ad5/F35-eGFP) expressing the enhanced green fluorescent protein gene in the presence or absence of CAR-SCF. Results Twenty-four hours after infection, more than 80% of M07e cells infected in the presence of CAR-SCF were eGFP-positive, compared with very few eGFP-positive cells following AdS-eGFP infection in the absence of CAR-SCF. The enhancement of Ad5-eGFP infection by CAR-SCF was greater than that caused by Ad5/F35-eGFP (50%). The ability of CAR-SCF to enhance Ad5-eGFP infectivity was highly dependent on cellular c-Kit expression levels. Furthermore, CAR-SCF also enhanced Ad5-mediated gene transfer into human primary CD34(+) cells. Conclusions The CAR-SCF fusion protein assists Ad5-mediated transduction to c-Kit(+) CAR(-) hematopoietic cells. The use of this fusion protein would enhance a utility of Ad5-mediated hematopoietic cell transduction strategies. Copyright (C) 2003 John Wiley Sons Ltd.
  • Takayuki Asano, Naohide Ageyama, Koichi Takeuchi, Mikio Momoeda, Yoshihiro Kitano, Kyoko Sasaki, Yasuji Ueda, Yutaka Suzuki, Yasushi Kondo, Ryuzo Torii, Mamoru Hasegawa, Shigeo Ookawara, Kiyonori Harii, Keiji Terao, Keiya Ozawa, Yutaka Hanazono
    Transplantation 76 (7) 1061 - 7 0041-1337 2003/10 [Refereed][Not invited]
    BACKGROUND: To achieve human embryonic stem (ES) cell-based transplantation therapies, allogeneic transplantation models of nonhuman primates would be useful. We have prepared cynomolgus ES cells genetically marked with the green fluorescent protein (GFP). The cells were transplanted into the allogeneic fetus, taking advantage of the fact that the fetus is so immunologically immature as not to induce immune responses to transplanted cells and that fetal tissue compartments are rapidly expanding and thus providing space for the engraftment. METHODS: Cynomolgus ES cells were genetically modified to express the GFP gene using a simian immunodeficiency viral vector or electroporation. These cells were transplanted in utero with ultrasound guidance into the cynomolgus fetus in the abdominal cavity (n=2) or liver (n=2) at the end of the first trimester. Three fetuses were delivered 1 month after transplantation, and the other, 3 months after transplantation. Fetal tissues were examined for transplanted cell progeny by quantitative polymerase chain reaction and in situ polymerase chain reaction of the GFP sequence. RESULTS: A fluorescent tumor, obviously derived from transplanted ES cells, was found in the thoracic cavity at 3 months after transplantation in one fetus. However, transplanted cell progeny were also detected (approximately 1%) without teratomas in multiple fetal tissues. The cells were solitary and indistinguishable from surrounding host cells. CONCLUSIONS: Transplanted cynomolgus ES cells can be engrafted in allogeneic fetuses. The cells will, however, form a tumor if they "leak" into an improper space such as the thoracic cavity.
  • Hiroaki Shibata, Yutaka Hanazono, Naohide Ageyama, Takeyuki Nagashima, Yasuji Ueda, Mamoru Hasegawa, Keiya Ozawa, Yasuhiro Yoshikawa, Keiji Terao
    American journal of primatology 61 (1) 3 - 12 0275-2565 2003/09 [Refereed][Not invited]
    Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.
  • T Kohno, H Mizukami, M Suzuki, Y Saga, Y Takei, M Shimpo, T Matsushita, T Okada, Y Hanazono, A Kume, Sato, I, K Ozawa, K Ozawa
    CANCER RESEARCH 63 (16) 5091 - 5094 0008-5472 2003/08 [Refereed][Not invited]
    Interleukin-10 (IL-10) is an immunosuppressive cytokine produced by T lymphocytes and drawing attention as an inhibitor of tumor angiogenesis. In this study, we investigated antiangiogenic and tumor suppressive effects of IL-10 in ovarian cancer cells. mIL-10-expressing phismid was transferred into two ovarian cancer cell lines, SHIN-3 [vascular endothelial growth factor (VEGF) producing] and KOC-2S (non-VEGF producing). After selection, mIL-10-expressing cells were obtained as SHIN-3/mIL-10 and KOC-2S/mIL-10. No significant differences were observed in in vitro growth properties between mIL-10-expressing cells and control (luciferase expressing) cells in either KOC-2S or SHIN-3. The angiogenic activities of mIL-10-expressing cells were measured by dorsal air sac assay, which detected the number of newly formed blood vessels within a chamber in vivo. In addition, tumor formation was evaluated by s.c. tumor transplantation, and survival was monitored after i.p. injection of ovarian cancer cells into BALB/c nude mice. Both in vivo angiogenic activity and tumor growth were significantly inhibited in SHIN-3/mIL-10 cells compared with the control. Moreover, peritoneal dissemination was inhibited, and the survival period was significantly prolonged (mean survival days >90 versus 36). In contrast, in the case of KOC-2S cells, no significant differences were observed in any of the parameters tested. These results indicate that IL-10 has suppressive effects on angiogenesis, tumor growth, and peritoneal dissemination of VEGF-producing ovarian cancer cells. Although the mechanisms of the antiangiogenic effect of IL-10 are still unclear, the potential usefulness of IL-10-mediated gene therapy of ovarian cancer was suggested.
  • M Kametaka, A Kum, T Okada, H Mizukami, Y Hanazono, K Ozawa
    CANCER SCIENCE 94 (7) 639 - 643 1347-9032 2003/07 [Refereed][Not invited]
    Allogeneic bone marrow transplantation and donor lymphocyte infusion are powerful treatments for chemotherapy-resistant leukemia. Tumor eradication is attributed to a graft-versus-leukemia reaction by the donor-derived cytotoxic T lymphocytes (CTLs), but the same cell population may cause severe graft-versus-host disease. One strategy to suppress harmful CTL activity is to incorporate a suicide gene into the donor lymphocytes prior to infusion, and to destroy these cells when they aggressively attack nonmalignant host tissues. In this study, we investigated the feasibility of using a Fas-estrogen receptor fusion protein (MfasER) to control T cell-mediated cytotoxicity, based on our previous finding that the chimera transmits a Fas-mediated death signal through activation by estrogen binding. A murine CTL line CTLL-2 was transfected with a vector encoding MfasER, and the growth, viability and cytotoxic activity of the transfected cells (CTLL/MfasER) were analyzed. The expression of apoptosis-related proteins such as Fas ligand and perforin was also investigated. In the absence of estrogen, CTLL/MfasER showed similar growth to parental CTLL-2, and the killing activity was preserved. Addition of 10(-7) M estrogen induced a rapid apoptosis of CTLL/MfasER, and the cytotoxicity was severely impaired. A decrease of Fas ligand and perforin in the estrogen-treated CTLL/MfasER was seen in an immunoblot analysis. These functional and biochemical analyses showed that the estrogen-inducible apoptosis in MfasER-expressing CTLs rapidly terminated their target cell killing. The feasibility of using the MfasER-estrogen system to control graft-versus-host disease was demonstrated.
  • T Nomoto, T Okada, K Shimazaki, H Mizukami, T Matsushita, Y Hanazono, A Kume, K Katsura, Y Katayama, K Ozawa
    NEUROSCIENCE LETTERS 340 (2) 153 - 157 0304-3940 2003/04 [Refereed][Not invited]
    Genetic modification of the gerbil hippocampal neuronal cells in vivo helps us understand the mechanisms of neuronal function under various circumstances such as ischemic insult. In this study, we examined the distinct distribution of the recombinant adeno-associated virus type 2 (rAAV2) and rAAV5 vectors for gene delivery to primary cultured cells and the gerbil hippocampus. Mixed cortical cultures containing both neurons and astrocytes from E17 rat embryos were infected with rAAVs containing the Cytomegalovirus virus (CMV) promoter. rAAV2 was preferably transduced to neurons, whereas rAAV5 was inclined to be transduced to astrocytes in vitro. rAAV2 and rAAV5 vectors, each with the CMV or Rous sarcoma virus (RSV) promoter, were injected into the gerbil hippocampus using a stereotaxic apparatus. Five days after injection, transgene expression was analyzed with X-gal staining. In the gerbil hippocampus, rAAV5 with the CMV promoter achieved, a higher overall transgene expression than rAAV2 with the CMV promoter. The transgene expression of rAAV2 with the RSV promoter was found in the pyramidal and granular cells, while the transgene expression of rAAV5 with the RSV promoter was preferentially found in the granular cells. These findings would be valuable in optimizing rAAV-mediated gene transfer to the gerbil hippocampus. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.
  • T Nomoto, T Okada, K Shimazaki, H Mizukami, T Matsushita, Y Hanazono, A Kume, K Katsura, Y Katayama, K Ozawa
    NEUROSCIENCE LETTERS 340 (2) 153 - 157 0304-3940 2003/04 [Refereed][Not invited]
    Genetic modification of the gerbil hippocampal neuronal cells in vivo helps us understand the mechanisms of neuronal function under various circumstances such as ischemic insult. In this study, we examined the distinct distribution of the recombinant adeno-associated virus type 2 (rAAV2) and rAAV5 vectors for gene delivery to primary cultured cells and the gerbil hippocampus. Mixed cortical cultures containing both neurons and astrocytes from E17 rat embryos were infected with rAAVs containing the Cytomegalovirus virus (CMV) promoter. rAAV2 was preferably transduced to neurons, whereas rAAV5 was inclined to be transduced to astrocytes in vitro. rAAV2 and rAAV5 vectors, each with the CMV or Rous sarcoma virus (RSV) promoter, were injected into the gerbil hippocampus using a stereotaxic apparatus. Five days after injection, transgene expression was analyzed with X-gal staining. In the gerbil hippocampus, rAAV5 with the CMV promoter achieved, a higher overall transgene expression than rAAV2 with the CMV promoter. The transgene expression of rAAV2 with the RSV promoter was found in the pyramidal and granular cells, while the transgene expression of rAAV5 with the RSV promoter was preferentially found in the granular cells. These findings would be valuable in optimizing rAAV-mediated gene transfer to the gerbil hippocampus. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.
  • Y Hanazono, T Asano, Y Ueda, K Ozawa
    TRENDS IN CARDIOVASCULAR MEDICINE 13 (3) 106 - 110 1050-1738 2003/04 [Refereed][Not invited]
    During the past several years, many articles have described how human embryonic stem (ES) cells and adult hematopoietic stem cells (HSCs) can differentiate into cardiac muscle, blood vessels, and various other types of cells. The articles raised the expectation that these stem cells may become useful for the treatment of a variety of diseases, including cardiovascular diseases. Genetic manipulation of ES cells and HSCs would be important for such future applications of the cells. Until now, retroviral vectors have been used primarily for stable expression of transgenes in murine ES cells and HSCs. Because murine models may not predict reliably the biology of ES cells and HSCs in humans, we have utilized primate ES cells and HSCs as targets of gene transfer. We have shown that primate ES cells and HSCs can be transduced efficiently with lentiviral vectors derived from the simian immunodeficiency virus, and that the high transgene expression persists without transcriptional silencing. This highly efficient gene transfer method allows for safe and faithful gene delivery to primate ES cells and HSCs to test potential research and therapeutic applications. (C) 2003, Elsevier Science Inc.
  • Takeyuki Nagashima, Yasuji Ueda, Yutaka Hanazono, Akihiro Kume, Hiroaki Shibata, Naohide Ageyama, Keiji Terao, Keiya Ozawa, Mamoru Hasegawa
    Biochemical and biophysical research communications 303 (1) 170 - 6 0006-291X 2003/03 [Refereed][Not invited]
    We previously developed "selective amplifier genes (SAGs)" which confer a growth advantage to transduced cells. The SAG is a chimeric gene encoding the G-CSF receptor (GCR) and the estrogen or tamoxifen (Tm) receptor and is able to expand transduced hematopoietic cells by treatment with estrogen or Tm. In the current study, we examined the in vitro efficacy of modified SAGs containing the thrombopoietin (TPO) receptor (c-Mpl) gene instead of GCR as a more potent signal generator. In addition, we constructed various mutant Mpl-type SAGs to abolish the responsiveness to endogenous TPO while retaining Tm-dependency. When Ba/F3 cells were retrovirally transduced with the Mpl-type SAGs, the cells showed Tm- and TPO-dependent growth even without IL-3. The Mpl-type SAGs induced more potent proliferation of Ba/F3 and cynomolgus CD34(+) cells than the GCR-type SAG. One mutant Mpl-type SAG (Delta GCRMplTmR) successfully lost the responsiveness to TPO without affecting the Tm-dependence.
  • Takeyuki Nagashima, Yasuji Ueda, Yutaka Hanazono, Akihiro Kume, Hiroaki Shibata, Naohide Ageyama, Keiji Terao, Keiya Ozawa, Mamoru Hasegawa
    Biochemical and biophysical research communications 303 (1) 170 - 6 0006-291X 2003/03 [Refereed][Not invited]
    We previously developed "selective amplifier genes (SAGs)" which confer a growth advantage to transduced cells. The SAG is a chimeric gene encoding the G-CSF receptor (GCR) and the estrogen or tamoxifen (Tm) receptor and is able to expand transduced hematopoietic cells by treatment with estrogen or Tm. In the current study, we examined the in vitro efficacy of modified SAGs containing the thrombopoietin (TPO) receptor (c-Mpl) gene instead of GCR as a more potent signal generator. In addition, we constructed various mutant Mpl-type SAGs to abolish the responsiveness to endogenous TPO while retaining Tm-dependency. When Ba/F3 cells were retrovirally transduced with the Mpl-type SAGs, the cells showed Tm- and TPO-dependent growth even without IL-3. The Mpl-type SAGs induced more potent proliferation of Ba/F3 and cynomolgus CD34(+) cells than the GCR-type SAG. One mutant Mpl-type SAG (Delta GCRMplTmR) successfully lost the responsiveness to TPO without affecting the Tm-dependence.
  • A Kume, M Koremoto, RF Xu, T Okada, H Mizukami, Y Hanazono, M Hasegawa, K Ozawa
    JOURNAL OF GENE MEDICINE 5 (3) 175 - 181 1099-498X 2003/03 [Refereed][Not invited]
    Background Hematopoietic stem-cell-directed gene transfer has achieved limited success in transducing clinically relevant levels of target cells. The expansion of gene-modified cells is one way to circumvent the problem of inefficient transduction with current vectors. To this end, we have developed 1 selective amplifier genes' (SAGS) that encode chimeric proteins that are a fusion of granulocyte colony-stimulating factor receptor and the steroid-binding domain. Prototype SAGS conferred estrogen-responsive growth on murine hematopoietic progenitors. Methods We constructed a retroviral vector coexpressing an SAG for 4-hydroxytamoxifen (Tm)-specific proliferation and the enhanced green fluorescent protein (EGFP). Murine bone marrow cells were transduced with this vector and transplanted into myeloablated mice. Subsequently, recipients were challenged with Tm, and EGFP(+) cells were enumerated. Results The challenge induced a significant increase in EGFP(+) leukocytes (21 +/- 4% to 27 +/- 5%), while EGFP(+) cells decreased in untreated animals (21 +/- 5% to 10 +/- 3%). Three months later, bone marrow cells were transplanted from the unchallenged mice to secondary hosts. Again the administration of Tm resulted in an increase of EGFP(+) cells (16 +/- 4% to 35 +/- 3%), contrasting to a decrease in controls (22 +/- 4% to 12 +/- 4%), and the difference was significant for more than 3 months. A detailed study of lineage showed a preferential expansion of EGFP(+) cells in granulocytes and monocytes following Tm administration. Conclusions Long-term repopulating cells were transduced with the SAG, and the transduced granulocyte/monocyte precursors were most likely to be expandable in vivo upon Tm stimulation. Copyright (C) 2002 John Wiley Sons, Ltd.
  • YY Lu, LJ Wang, S Muramatsu, K Ikeguchi, K Fujimoto, T Okada, H Mizukami, T Matsushita, Y Hanazono, A Kume, T Nagatsu, K Ozawa, Nakano, I
    NEUROSCIENCE RESEARCH 45 (1) 33 - 40 0168-0102 2003/01 [Refereed][Not invited]
    Adeno-associated virus (AAV) vector has been developed as an attractive gene delivery system with proven safety. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for amyotrophic lateral sclerosis (ALS) and other motor neuron diseases. The purpose of this report was to investigate transgenic GDNF expression at different time points post AAV mediated GDNF intramuscular delivery. An AAV vector was constructed to encode a recombinant fusion of GDNF tagged with a FLAG sequence at the C-terminal (AAV-GDNF) to distinguish it from its endogenous counterpart. A single intramuscular injection of AAV-GDNF led to substantial expression of transgenic GDNF which remained for at least 10 months in transduced gastrocnemius muscle. This transgenic GDNF was distributed in a large number of myofibers, mainly in the vicinity of the sarcolemma and predominantly concentrated at the sites of neuromuscular junctions (NMJs). Furthermore, transgenic GDNF, but not P-galactosidase expressed as a control, was detected in the motoneurons that projected axons to the injected muscles, thus, indicating retrograde axonal transportation of the transgenic GDNF. This study provides a basis for a strategy of intramuscular AAV-GDNF delivery to protect motoneurons as a possible means of ALS treatment. (C) 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Muramatsu S, Wang L, Ikeguchi K, Fujimoto K, Nakano I, Okada T, Mizukami H, Hanazono Y, Kume A, Nakano I, Ozawa K
    International review of neurobiology 55 205 - 222 0074-7742 2003 [Refereed][Not invited]
  • A Kume, Y Hanazono, H Mizukami, T Okada, K Ozawa
    INTERNATIONAL JOURNAL OF HEMATOLOGY 76 (4) 299 - 304 0925-5710 2002/11 [Refereed][Not invited]
    Although gene transfer into hematopoietic stem cells holds a considerable therapeutic potential, clinical trials targeting this cell compartment have achieved limited success. Poor transduction efficiency with gene transfer vectors used in human studies has hindered delivering therapeutic genes to clinically relevant numbers of target cells. One way to overcome the low-efficiency problem is by selecting or expanding the number of genetically modified cells to a suprathreshold level to achieve clinical efficacy. This approach may be further classified into 2 categories: one is to transfer a drug resistance gene and eliminate unmodified cells with cytotoxic drugs, and the other is to confer a direct growth advantage on target cells. This review aims at an overview of recent advances involving these strategies, with some details of "selective amplifier genes," a novel system that we have developed for specific expansion of genetically modified hematopoietic cells.
  • Naohide Ageyama, Yutaka Hanazono, Hiroaki Shibata, Keiko Ohto, Fumiko Ono, Takeyuki Nagashima, Yasuji Ueda, Robert E Donahue, Mamoru Hasegawa, Keiya Ozawa, Yasuhiro Yoshikawa, Keiji Terao
    Comparative medicine 52 (5) 445 - 51 1532-0820 2002/10 [Refereed][Not invited]
    We have established safe and efficient methods for autologous hematopoietic stem cell (HSC) transplantation in cynomolgus monkeys (Macaca fascicularis) that include regimens of supportive care to ensure survival during hematopoietic reconstitution following otherwise lethal total body irradiation. Eleven young adult cynomolgus monkeys were studied. Bone marrow was aspirated from the ilium and/or tuber ischiae after administration of recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF). Using the immunomagnetic selection method, CD34+ cells were then isolated (90 to 95% pure) as a fraction containing HSCs. Just prior to transplantation, the animals received myeloablative total body irradiation-500 to 550 cGy daily for two days. The monkeys re-infused with CD34+ cells developed moderate to severe myelosuppression, with some animals requiring intravenous hyperalimentation, antibiotic administration, and blood transfusion. Hematopoiesis was restored in all animals after transplantation. It took 12 days, on average, until the peripheral white blood cell count reached more than 1,000 cells/microl. Up to two years after transplantation, signs of radiation-induced pneumonitis or other radiation-related disorders were not evident at the aforementioned dose of irradiation. This transplantation model will be useful for testing new approaches using HSCs for therapy of many diseases and will offer unique insights into the biology of these cells.
  • Okada T, Nomoto T, Shimazaki K, Lijun W, Lu Y, Matsushita T, Mizukami H, Urabe M, Hanazono Y, Kume A, Muramatsu S, Nakano I, Ozawa K
    Methods (San Diego, Calif.) 2 28 (2) 237 - 247 1046-2023 2002/10 [Refereed][Not invited]
  • Y Hanazono, K Terao, H Shibata, T Nagashima, N Ageyama, T Asano, Y Ueda, Kato, I, A Kume, M Hasegawa, K Ozawa
    JOURNAL OF GENE MEDICINE 4 (5) 470 - 477 1099-498X 2002/09 [Refereed][Not invited]
    Background The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning. Methods Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34(+) cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy x 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells. Results Although 13-37% of transduced cells contained the GFP provirus and 11-13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5-10%) of clonogenic progenitors contained the provirus and fluoresced exvivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes. Conclusions Low levels of GFP-transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP this block an treatment ex vivo and in vivo. Copyright (C) 2002 John Wiley Sons, Ltd.
  • T Asano, Y Hanazono, Y Ueda, S Muramatsu, A Kume, H Suemori, Y Suzuki, Y Kondo, K Harii, M Hasegawa, N Nakatsuji, K Ozawa
    MOLECULAR THERAPY 6 (2) 162 - 168 1525-0016 2002/08 [Refereed][Not invited]
    The ability to stably introduce genetic material into primate embryonic stem (ES) cells could allow their broader application. We previously derived ES cell lines from cynomolgus monkey blastocysts. In this study, we examined lentiviral gene transfer into cynomolgus ES cells. When cynomolgus ES cells were transduced once with a simian immunodeficiency virus (SIV)-based lentivirus vector encoding the green fluorescent protein (GFP) gene, most cells (around 90%) fluoresced, and high levels of GFP expression persisted for 5 months without selection procedures. In addition, high levels of GFP expression were observed during embryoid body formation. On the other hand, transduction of mouse ES cells with the SIV-based vector resulted in lower gene transfer rates, implying that SIV vectors can transduce primate ES cells more efficiently than mouse ES cells. The use of GFP as a reporter gene allows direct and simple detection of successfully transduced ES cells and facilitates monitoring of ES cell proliferation and differentiation both in vitro and potentially in vivo. Furthermore, this highly efficient gene transfer method allows faithful gene delivery to primate ES cells with potential for both research and therapeutic application.
  • Wang LJ, Lu YY, Muramatsu S, Ikeguchi K, Fujimoto K, Okada T, Mizukami H, Matsushita T, Hanazono Y, Kume A, Nagatsu T, Ozawa K, Nakano I
    The Journal of neuroscience : the official journal of the Society for Neuroscience 16 22 (16) 6920 - 6928 0270-6474 2002/08 [Refereed][Not invited]
  • Okada T, Shimazaki K, Nomoto T, Matsushita T, Mizukami H, Urabe M, Hanazono Y, Kume A, Tobita K, Ozawa K, Kawai N
    Methods in enzymology 346 378 - 393 0076-6879 2002 [Refereed][Not invited]


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