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SHIGERU MIYAKI

  • Faculty of Medicine
  • School of Medicine
  • Professor
Last Updated :2026/08/25

Researcher Information

Degree

  • Ph.D.（2004/03 University of Tsukuba）

Research funding number

  • 10392490

ORCID ID

J-Global ID

Profile

  • Currently I am working on a project concerning the expression and function of micro RNAs (miRNAs) in osteoarthritis. This project is highly original as it is the first to address this new class of regulatory RNAs in joint tissue. I have a strong knowledge base in cell and molecular biology and is familiar with a broad range of experimental approaches and models in this field. I have generated mutant mice, established animal models, I am an expert molecular and cell biologist and had in-depth expertise in bioinformatics. My knowledge base not only includes skeletal biology but acquire insight into mechanisms of pathogenesis in musculoskeletal diseases. The goal of proposed studies is to reveal that exosome-mediated extracellular miRNA transfer in joint cell signaling and osteoarthritis pathogenesis. The studies have the potential to reveal important new regulatory pathways in osteoarthritis pathogenesis, and open new insights on osteoarthritis mechanisms. This may lead to the identification of novel therapeutic targets and biomarkers for diagnosis in osteoarthritis. Furthermore, this proposal would not only pave the pathway for understanding and treating osteoarthritis, but it also has the potential to open doors to understanding the general role of extracellular miRNAs as a signaling molecule pathway in a complex network under various physiological and pathophysiological events.

Research Interests

  • Musculoskeletal Biology, Osteoarthritis, miRNAs, Exosomes, Regenerative Medicine, Tendon, Ligament   

Research Areas

  • Life sciences / Orthopedics

Academic & Professional Experience

  • 2025/05 - Today  Kagawa UniversityFaculty of Medicine教授
  • 2023/06 - 2025/05  Hiroshima University病院 未来医療センターProfessor (Special recognition)
  • 2011/03 - 2023/06  Hiroshima UniversityHospitalAssociate professor
  • 2009/06 - 2011/03  The Scripps Research InstituteSr. Research Accosiate
  • 2007/07 - 2009/05  The Scripps Research InstituteResearch Associate
  • 2004/03 - 2007/06  National Center for Child Health and DevelopmentResearch Associate

Association Memberships

  • 日本分子生物学会   日本解剖学会   老化促進モデルマウス（SAM）学会   The Japanese Society for Bone and Mineral Research   The Japanese society for regenerative medicine   The Japanese Society of Cartilage Metabolism   Orthopaedic Research Society   Osteoarthritis research society international   

Published Papers

MISC

  • TumdCreノックインマウスの作製とTnmd陽性細胞の系譜解析
    山家 新勢; 吉本 由紀; 樋口 真之輔; 味八木 茂; 宿南 知佐  日本骨代謝学会学術集会プログラム抄録集  39回-  143  -143  2021/10
  • 変形性膝関節症マウスモデルを用いた軟骨下骨の組織学的スコアリングシステムの確立
    柳樂 慶太; 味八木 茂; 生田 祥也; 眞田 洋平; 篠原 正浩; 安達 伸生; 永島 英樹; Lotz Martin  日本整形外科学会雑誌  93-  (8)  S1949  -S1949  2019/09
  • 変形性膝関節症マウスモデルを用いた軟骨下骨の組織学的スコアリングシステムの確立
    柳樂 慶太; 真田 洋平; 篠原 正浩; 萩野 浩; 味八木 茂  日本骨代謝学会学術集会プログラム抄録集  37回-  229  -229  2019/09
  • ホメオボックス遺伝子Mkx欠損マウスの骨格筋表現型
    秋本 崇之; 狩野 豊; 中村 一隆; 味八木 茂; 浅原 弘嗣; 牛田 多加志  体力科学  64-  (6)  560  -560  2015/12
  • Stf-083010, the Inhibitor of ER Stress Transducer IRE1, Suppresses Rheumatoid Synovitis
    Soutarou Izumi; Tomoyuki Nakasa; Shigeru Miyaki; Mitsuo Ochi  ARTHRITIS & RHEUMATOLOGY  67-  2015/10
  • Bach1ノックアウトマウスは加齢性および実験的変形性関節症の症状を軽減する
    石飛 博之; 味八木 茂; 高田 剛志; 中佐 智幸; 五十嵐 和彦; 越智 光夫  日本抗加齢医学会総会プログラム・抄録集  15回-  229  -229  2015/05
  • Bach1ノックアウトマウスは加齢性および実験的変形性関節症の症状を軽減する
    高田 剛志; 味八木 茂; 石飛 博之; 中佐 智幸; 五十嵐 和彦; 越智 光夫  日本整形外科学会雑誌  88-  (8)  S1617  -S1617  2014/08
  • Bach1ノックアウトマウスはHO-1の発現により変形性膝関節症の進行を予防する
    高田 剛志; 味八木 茂; 石飛 博之; 中邑 祥博; 加藤 智弘; 五十嵐 和彦; 越智 光夫  日本整形外科学会雑誌  87-  (8)  S1398  -S1398  2013/08
  • CARTILAGE-SPECIFIC MICRORNA-140 REGULATES TISSUE HOMEOSTASIS AND PROTECTS AGAINST OSTEOARTHRITIS-LIKE PATHOLOGY
    S. Miyaki; T. Sato; A. Inoue; S. Otsuki; Y. Ito; S. Yokoyama; Y. Kato; S. Yamashita; T. Nakasa; M. K. Lotz; H. Kudo-Ueno; H. Asahara  OSTEOARTHRITIS AND CARTILAGE  18-  S37  -S37  2010/10
  • ストレス誘導性miR-23aはatrogin-1の転写後制御を介して骨格筋萎縮を抑制する
    和田 正吾; 奥津 光晴; 味八木 茂; 鈴木 克彦; 牛田 多加志; 秋本 崇之  体力科学  57-  (6)  674  -674  2008/12
  • 94. メカニカルストレス/筋収縮によるPGC-1αの転写制御(運動器,一般口演,第63回日本体力医学会大会)
    秋本 崇之; 和田 正吾; 相澤 勝治; 味八木 茂; 牛田 多加志  体力科學  57-  (6)  679  -679  2008/12
  • Inhibition of osteoclastogenesis by over expression of microrna-223 in rheumatoid arthritis
    Hayatoshi Shibuya; Tomoyuki Nakasa; Shigeru Miyaki; Masataka Deie; Osami Suzuki; Hiroshi Asahara; Mitsuo Ochi  ARTHRITIS AND RHEUMATISM  58-  (9)  S645  -S645  2008/09
  • Expression of mir-140 in articular and during chondrogenesis
    Shigeru Miyaki; Tomoyuki Nakasa; Atsushi Inoue; Shawn P. Grogan; Kenta Uchibe; Reiji Higashiyama; Martin K. Lotz; Hiroshi Asahara  ARTHRITIS AND RHEUMATISM  58-  (9)  S648  -S648  2008/09
  • 105. 膝関節内靭帯に発現する遺伝子の網羅的解析(運動器,第62回日本体力医学会大会)
    秋本 崇之; 槌谷 宏平; 味八木 茂; 浅原 弘嗣; 牛田 多加志  体力科學  56-  (6)  631  -631  2007/12
  • T Akimoto; T Uchida; S Miyaki; H Akaogi; T Tateishi; T Fukubayashi  MATERIALS SCIENCE & ENGINEERING C-BIOMIMETIC AND SUPRAMOLECULAR SYSTEMS  24-  (3)  387  -389  2004/04

Industrial Property Rights

  • 6762007:変形性関節症予防用組成物、変形性関節症予防用食品組成物、変形性関節症予防用食品添加物及び変形性関節症予防用医薬  

Awards & Honors

  • 2010 Arthritis National Research Foundation, Grant Award
  • 2010 World Congress on Osteoarthritis (OARSI), Young Investigator Award
  • 2010 Orthopaedic Research Society (USA), The New Investigator Recognition Award
  • 2009 Arthritis National Research Foundation (USA), Grant Award

Research Grants & Projects

  • 椎間板幹・前駆細胞におけるmiR-23による恒常性維持機構の解明
    日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2025/04 -2028/03 
    Author : 滝本 晶; 味八木 茂
  • 人工赤血球を用いた組織灌流保存法の開発
    日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2025/04 -2028/03 
    Author : 四宮 陸雄; 酒井 宏水; 味八木 茂; 中佐 智幸
  • レチノイン酸シグナルによる軟骨細胞の分化段階に応じた制御機構の解明
    日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2025/04 -2028/03 
    Author : 内部 健太; 味八木 茂; 宿南 知佐
  • Creation of data-driven chemical reprogramming technologies by experiment optimization AI
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2024/06 -2027/03 
    Author : 山西 芳裕; 味八木 茂; 鈴木 淳史
     
    iPS細胞を介さずに目的の臓器の細胞に直接変換するダイレクトリプログラミング(Direct Reprograming (DR)）が革新的な再生医療技術として注目されているが、転写因子の遺伝子導入による標準的方法は腫瘍化のリスクがある。本研究では、転写因子の遺伝子導入だけでなく、承認薬や食品成分などの化合物によるDR（ケミカルリプログラミング）を提唱する。その実現に向けて、低分子化合物添加に応答するオミックスデータや分子構造データを解析し、細胞変換を誘導する低分子化合物群やそれらの最適な組み合わせを予測するAIを開発する。低分子化合物の濃度や時間点などの実験条件を区別し、最適な実験条件の組み合わせを見つける最適化アルゴリズムを開発する。さまざまな細胞変換を担うと予測された低分子化合物を実験検証し、効果的な低分子化合物セットを同定する。 まず、転写因子や低分子化合物に関する多階層オミックスデータを整備した。特に、転写因子や低分子化合物がヒト細胞に与える影響を評価するため、ヒト細胞における遺伝子発現プロファイルを解析した。転写因子の遺伝子導入や低分子化合物の添加に関するトランスクリプトームデータは、GEOから得た。転写因子の情報は、ChipAtlas, BRENDA, KEGGなどのデータベースから得た。低分子化合物の濃度や時間点、細胞の違いにおける遺伝子発現パターンのばらつきを解析した。転写因子の遺伝子導入における時間点、細胞の違いにおける遺伝子発現パターンのばらつきを解析した。ダイレクトリプログラミングを誘導することが報告されている低分子化合物の情報を収集して、その作用機序を標的タンパク質や標的パスウェイなどの視点から推定した。学会発表も口頭発表を1件行った。
  • 系統追跡解析を用いた腱板の恒常性維持と治癒における細胞の動態と役割の解析
    日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2024/04 -2027/03 
    Author : 原田 洋平; 味八木 茂
     
    本研究の目的は、CreERT2-tdTomatoマウスを用いて、腱板の成熟・加齢および損傷における腱細胞・腱前駆細胞・軟骨細胞の動態と役割を解明することである。 まずはScleraxis（Scx）CreERT2-tdTomatoマウスを作製し、Scx陽性細胞の分布および加齢による変化を解析した。3週齢（未成熟齢：成長期）および9週齢（成熟齢：成獣期）のマウスにタモキシフェン（TAM）を投与することでその時点での陽性細胞を標識し、2週および1年後のScx陽性細胞の分布を解析した。その結果、腱実質部では、成長期および成熟期のいずれにおいても、TAM投与後2週時点でScx陽性細胞の割合が高く、1年後には減少しており、変化率は成長期でより顕著であった。一方、腱付着部ではいずれの時期でもScx陽性細胞の割合が低く、時間経過による大きな変化はなかった。腱実質部でのScx陽性細胞は、成長期での変化率が大きいことから、腱の成熟に関与している可能性が示唆された。 また、10週齢のScx CreERT2-tdTomatoマウスを用い、腱板損傷モデルを作製し、腱板損傷に伴うScx陽性細胞の変化を解析した。腱板損傷前にTAMで標識したモデルでは、損傷部および瘢痕部にScx陽性細胞の集積が認められたが、腱板損傷後にTAMで標識したモデルでは、腱板損傷部に集積を認めなかった。以上より、腱内在Scx陽性細胞は損傷部に集積せず、成体の腱板修復には関与しない可能性が示唆された。
  • Investigation of Novel Therapeutic Approaches for Osteonecrosis of the femoral head
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2024/04 -2027/03 
    Author : 庄司 剛士; 味八木 茂; 中佐 智幸
     
    本研究では、複数の遺伝子発現を同時に制御する疾患特異的/修飾的なmicroRNA(miRNA)投与を行うことにより、壊死骨・骨壊死周辺領域の血管形成・骨形成促進作用を介した早期骨修復を促進し、また併せてmiRNAによる遺伝子制御を介した関節内の恒常性維持を図ることで、大腿骨頭の圧潰、また病期進行を抑制する効果が得られることを仮説とし研究を進めている。これまで、大腿骨頭壊死症(ONFH)患者由来の関節液中のmiRNAの発現プロファイルを解析し、ONFHの関節液中では病期、ONFH関連、年齢等に関わらず、miR-210, miR-296, miR-659, let-7cの優位な発現の亢進を認めた。また、ラット骨壊死症モデルを用いたhydroxyapatiteをScaffoldとしたmiRNA-31/210の局所投与群においては、microCT解析において、有意に骨量(BV/TV)と骨梁数の増加を認め、また組織学的評価においても、新生骨形成と血管新生が最も顕著であり、HAの周囲に緻密な新生骨の形成が観察された。毛細血管密度はmiRNA-31/210miR-31の局所投与群において(35.17 vessels//mm2)controlと比較し(12.15 vessels//mm2)有意に高値を示した。
  • 腸を中心とした臓器間ネットワークからの抗老化機能性食品の作用機序の解明
    日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2024/04 -2027/03 
    Author : 松原 主典; 味八木 茂
     
    腸の老化が全身の老化につながることが明らかになってきている。また，腸は他の臓器とネットワークを形成しており，腸の老化がそのネットワークの破綻につながり，各種臓器，そして全身の老化につながると考えられている。抗老化効果を示す機能性食品の作用機序も，この腸を中心とする臓器間ネットワークと関連している可能性がある。本研究ではこの作用機序について検討を進める。 老化の研究には，主に老化促進マウス（SAMP8）を使用しており，抗老化効果を示す機能性食品の腸に対する効果を検討するため，このマウスの加齢による腸の変化を検討している。腸管内分泌細胞について検討した結果，ペプチドYY産生細胞は加齢に伴って減少することが明らかとなった。一方，セロトニン産生細胞については，加齢による明確な変化は認められなかった。また，ソマトスタチン産生細胞やGLP-1産生細胞についてはさらに検討が必要である。パーキンソン病との関係で注目されているドーパミンレセプターD2については，SAMP8においても加齢による変化が起きることが示唆される結果が得られた。また，他系統のマウスと同様に，腸管の形態そのものも加齢によって変化することが確認できた。加齢によって変化する腸管内分泌細胞やドーパミンレセプターが抗老化効果を示す機能性食品の影響を受ける可能性が示唆されたことから，これらの細胞や分子を指標に研究を進めることにより，抗老化効果を示す機能性食品の作用機序解明につながることが期待できる。 発酵食品の抗老化効果が注目されており，特にヨーグルトや納豆に含まれるポリアミンが注目されている。そこで，発酵食品に多く含まれ，ポリアミン合成にも関わる機能成分の抗老化効果をSAMP8への投与で検討した。一定の抗老化効果は確認できたが，その有用性は加齢に伴って変化したことから，代謝物を含めて検討する必要があると考えられた。
  • 骨粗鬆症を介した腰椎椎体終板障害の機能解明および新たな治療戦略の確立
    日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2024/04 -2027/03 
    Author : 中前 稔生; 味八木 茂; 亀井 直輔
     
    超高齢社会である我が国において、腰痛の病態解明は喫緊の課題である。我々はこれまでに臨床研究において腰椎椎体終板障害が高齢者の腰痛に強く関与していることを明らかにしてきた。しかしながら、骨粗鬆症などによる椎体の骨密度低下による椎体終板障害に対する影響については未解明な部分が多い。そこで本研究では、椎体終板障害に対する骨粗鬆症の関与について、マウスモデルを用いて明らかにする。 まずは野生型マウスの自然経過について、腰椎の軟骨終板の組織学的評価を行った。それによると、終板の変化は生後3か月から生じ、以後終板の成熟が進行した。軟骨終板においては、軟骨内に小さな骨化を生じ、順次骨化が増大した。これらの軟骨終板の組織学的評価は独自のスコアリングを用いて評価した。 続いて老化促進マウスであるSAMP8を用いて椎体終板の評価を行った。まず、SAMP8ではコントロールであるSAMR1と比べてBMD (bone mineral density) が有意に低値であり、骨粗鬆症マウスとして適切であることを確認した。SAMP8において椎間板組織学的スコアリング（Masuda K, Spine 2005）を検討したところ、SAMR1と比較し有意差を認めなかった。軟骨終板の組織学的スコアリングを行ったところ、椎間板の末梢側の軟骨終板ではSAMP8とSAMR1では有意差を認めなかったが、中枢側の軟骨終板ではSAMR1に比較しSAMP8では有意にスコアリングが低値であり、軟骨終板の成熟は抑制されていた。ただし、検体数が少なく、現在随時検体数を増やし組織学的評価を継続中である。
  • Elucidation of the Functional Role of MicroRNA Clusters in the Myelination of the Central Nervous System
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2024/04 -2027/03 
    Author : 亀井 直輔; 味八木 茂; 中前 稔生
     
    マイクロリボ核酸（miRNA）のうち、miR-23a/bクラスターを構成するmiR-23aとmiR-27aは、中枢神経系におけるミエリン化において異なる役割を果たすことが知られています。しかし、同じクラスター内のmiRNAが補完的に機能する可能性が指摘されているため、クラスター特異的なmiRNAの機能は依然として議論の的となっている。 本研究では、中枢神経の髄鞘特異的にmiR-23a/bクラスターをノックダウンされるマウスし，空間的、時間的に限定したmiR-23a/bクラスターの欠損が髄鞘形成に与える影響を明らかにすることとした。 タモシキフェン投与によりproteolipid protein (PLP) を発現する髄鞘特異的にmiR-23a/bクラスターを欠損させるコンディショナルノックアウトマウス (Plp CreERT2;miR-23a/b cluster) を作製した。生後7日齢あるいは10週齢でタモキシフェンを投与し, それぞれ生後10週齢と12ヶ月齢でのmiR-23a/bクラスター欠損の影響を解析した。またタモキシフェンを投与しなかったマウスをコントロールとして用いた。 コントロールマウスと比較してHanging wire testとBalance beam testにおける運動機能の低下が認められた。また, 電子顕微鏡検査とLuxol Fast Blue染色により, KOマウスの脳と脊髄における髄鞘および脳脊髄白質の形成不全が明らかになった。 これらの結果は、miR-23a/bクラスターが成長と老化におけるミエリン化に重要な影響を与えることを示している。
  • Development of a novel cartilage tissue evaluation system using color information
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2023/04 -2026/03 
    Author : 石川 正和; 渡邉 朋信; 味八木 茂
     
    今年度は分光色差計を用いた軟骨組織の色情報の取得技術と組織切片との比較検討技術を確立するために、比較的容易にサンプルが入手できる、関節鏡視下半月部分切除術時に摘出したヒト半月板組織を使用した（男性10例，女性7例）。色情報として採取した組織片の表面から購入した分光色差計（日本電色工業株式会社製 SE7700）によりL*a*b*値を測定している。また組織を固定後，組織切片を作製し、サフラニンOファストグリン染色及びヘマトキシリンエオジン染色を行い、modified Pauli scoreにより組織学的評価を行った。得られた数値を用いて、色情報と組織の変性度の関連性を調査した。結果として、年齢と組織の変性の程度は正の相関を示していた。また色情報ではL*，a*, b*ともに組織の変性の程度と相関を示したが、臨床の現場で経験しているように、特に黄色味を表すb*が組織変性の程度と最も強い正の相関を示した。さらに、冷凍保存していたサンプルを用いて、グリコサミノグリカン(GAG)の含有量を計測した。GAG含有量と色情報の関係に関しては黄色味を示すb*のみで負の相関を示すことが明らかとなった。以上の結果から、分光色差計によって得られる半月板組織の色情報と組織の変性程度が相関することが明らかとなった。また、半月板組織における重要な糖タンパクの一つであるGAGの低下も色情報から予測できる可能性が示され、色情報の有用性が示されつつあると考えている。
  • miRNAを含むMSC-細胞外小胞による腱修復機構を基盤にした新規治療法の開発
    日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2023/04 -2026/03 
    Author : 安達 伸生; 味八木 茂; 亀井 直輔; 中佐 智幸; 宿南 知佐; 河崎 陸
     
    腱や靭帯は、運動機能の中心的役割を担うが、分子生物学的な情報は他の運動器に比べて極めて少なく、その治癒を促進する治療法は未だ開発されていない。昨年度までに、遺伝子発現制御ネットワークに重要な因子であるmicroRNA（miRNA）の生成に重要なプロセッシング酵素Dicerを腱・靭帯特異的にノックアウトしたマウスが腱の低形成を示すことを報告した。そして、この表現系に関与する腱組織で高発現しているmiRNAを同定した。本年度は、このmiRNAのノックアウトマウス作製を行った。このノックアウトマウスは、発生過程に大きな異常なく正常に生まれてきており、肉眼的には異常は認められていない。現在、解析に使用するために繁殖中であり、次年度よりノックアウトマウスにおけるアキレス腱の組織学的解析や損傷修復過程における影響について詳細な解析を行う。また、アキレス腱損傷モデルマウスにmiRNAを含む間葉系幹細胞（MSC）-細胞外小胞（EV）を局所投与することで、その治療効果を得られることを明らかにしたが、さらに治療効果を高めるためにコレステロールを末端に修飾したポリエチレングリコールを合成し、それを用いたゲル化EV作製を試みた。そして、このゲル化EVのインジェクタブル性、EVの徐放能を確認し、このゲルから放出されたEVが培養細胞へと取り込まれることを確認した。次年度より、このゲル化MSC-EVをアキレス腱損傷モデルマウスへと投与し、その治療効果について評価を行う予定である。今後、同定したmiRNAを豊富に含んだゲル化MSC-EVをアキレス腱損傷モデルマウスに局所投与し、その治療効果を明らかにするとともに、同定miRNAの腱発生・成熟や修復への役割を遺伝子改変マウスにより明らかにしていく。
  • Novel treatment strategy for cartilage defect in osteoarthritis using osteophyte cartilage
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2022/04 -2025/03 
    Author : Tomoyuki Nakasa
     
    Osteoarthritis (OA) is a common joint disease involving cartilage degeneration, causing pain and reduced function. Minced cartilage implantation (MCI) is a promising one-step treatment for large cartilage defects, but sourcing chondrocytes is difficult in advanced OA due to limited healthy cartilage. We evaluated osteophyte cartilage, common in advanced OA, as an alternative chondrocyte source. Samples from 22 knee arthroplasty patients were analyzed. Using cartilage fragment/atelocollagen composites, we assessed chondrocyte migration and proliferation.Histology showed osteophyte cartilage had higher type X collagen and greater cell proliferation. While gene expression profiles differed, key cartilage-related genes (COL2A1, ACAN, SOX9) were similarly expressed. Osteophyte chondrocytes also showed high glycosaminoglycan production. These findings suggest osteophyte cartilage may be a feasible chondrocyte source for MCI in treating OA-related cartilage defects.
  • Development of Osteosarcoma Therapy Using Prodrugs Capable of Drug Release Action Only Under Hypoxia
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2022/04 -2025/03 
    Author : FURUTA Taisuke
     
    In recent years, the development of anticancer drug treatments for malignant tumors of the limbs has led to improvements in prognosis, but there are still many issues to be addressed, such as treatment resistance and serious side effects. We have focused on hypoxia-activated prodrugs (HAPs), which are prodrugs that can only be released and exert their effects under hypoxic conditions, by attaching 2-nitroimidazole (NIM) to classical anticancer drugs. Prodrugs can exert pharmacological effects in hypoxic areas of malignant tumors, enhancing therapeutic effects, and can also be expected to reduce side effects by reducing pharmacological effects under normal oxygen conditions. Doxorubicin, the most important drug for osteosarcoma, is problematic due to its side effects, particularly cardiotoxicity. We will verify the improvement of treatment resistance and reduction of side effects using doxorubicin prodrug.
  • Mechanisms for the regulation of bone formation and maintenance by retinoic acid and their development into clinical applications.
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2022/04 -2025/03 
    Author : Uchibe Kenta
     
    The present study aimed to elucidate the regulatory mechanisms of retinoic acid signaling in the bone-associated cells at the molecular and individual level, as well as its application in the treatment of related diseases such as bone fractures. The results revealed that RARγ, a retinoic acid receptor, plays a certain role in the differentiation process from undifferentiated cells to osteoblasts. It was found that future investigation, such as spatial analysis, is needed at the individual level, as there is also an effect on the neighboring cartilage.
  • Identification of Skeletal Stem Cells Involved in the Homeostasis and Regeneration of Articular Cartilage
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2022/06 -2024/03 
    Author : Adachi Nobuo
     
    Osteoarthritis (OA) is a common musculoskeletal disease that affects approximately 20 million patients in Japan. Despite its prevalence, the pathogenesis of OA remains unclear, and no fundamental treatments or preventive measures have been established. Understanding homeostasis maintenance and repair mechanisms of joint tissues, including cartilage, is important for developing new treatments. This study aimed to identify skeletal stem cells and cartilage progenitor cells involved in joint maintenance and repair. To achieve this, mice capable of undergoing cell lineage analysis were created, allowing for the clarification of the roles of these cells by selectively removing them. The findings suggest the presence of candidate skeletal stem cells and cartilage progenitor cells in joint tissues. These results will pave the way for further studies focused on developing new OA treatments and cartilage regeneration strategies using these mice.
  • Creation of data-driven direct reprogramming by AI and avoidance of tumorigenic risk
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2021/07 -2024/03 
    Author : Yamanishi Yoshihiro
     
    Direct reprogramming, direct cell conversion without the use of iPS cells, is attracting attention as an innovative regenerative medicine technology. However, it is extremely difficult to identify the set of factors (e.g., transcription factors, small molecule compounds) that induce direct reprogramming. In this study, we developed information technologies to predict the transcription factors and small molecule compounds that induce direct reprogramming. Based on various multi-level omics data, we developed an optimization algorithm to predict the set of transcription factors that induce direct cell conversion and the set of small molecule compounds that replace them. We demonstrated the usefulness of this algorithm in the direct conversion of skin fibroblasts into neurons, cardiomyocytes, etc.
  • Functional elucidation of lumbar vertebral endplate disorders via microRNA mediation and establishment of novel therapeutic strategies
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2021/04 -2024/03 
    Author : Nakamae Toshio
     
    In the current study, we used knockout mice for microRNAs (miRNAs) that regulate gene expression networks to analyze the function of miRNAs in cartilage endplate degeneration. We evaluated the cartilage endplates using knockout mice for two highly expressed miRNAs in cartilage (miR-23a/b cluster and miR-26a). Histological findings showed no significant difference in cartilage endplate changes compared to controls in miR-26a knockout mice, whereas in miR-23a/b cluster knockout mice, the maturation of cartilage endplates was inhibited. Moreover, in histological evaluations of intervertebral discs, progression of intervertebral disc degeneration was not observed in miR-26a knockout mice, but it was observed early on in knockout mice for the miR-23a/b cluster.
  • Development of Ex-vivo Perfusion Therapy with Artificial Oxygen Carrier for Amputated Limb Preservation
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2021/04 -2024/03 
    Author : Rikuo Shinomiya
     
    When limb amputation occurs, the muscle tissue has poor tolerance to ischemia, and immediate revascularization surgery is necessary to prevent severe functional impairment. It is critical to develop methods to preserve the amputated limb until revascularization surgery. Basic research has explored ex-vivo perfusion therapy using blood to extend the preservation time of amputated limbs. However, allogeneic blood transfusion faces challenges such as blood type compatibility, infection risk, and supply problems. Artificial oxygen carrier was developed to solve these problems. In this study, we established the optimal perfusion conditions using an ex-vivo rat hind-limb perfusion model with blood. This protocol can further elucidate the efficacy of artificial oxygen carrier, which are being researched for clinical application in Japan.
  • Search and design of compounds with synergistic effects by AI
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Transformative Research Areas (B)
    Date (from‐to) : 2020/10 -2023/03 
    Author : Yamanishi Yoshihiro
     
    Chemotherapy that utilizes the synergistic effect of a combination of multiple drugs (drug synergy) has been recognized as an effective method for treatment of multifactorial diseases such as cancer and neurodegenerative diseases. This research group develops statistical methods for predicting drug combinationss with synergistic effects, and machine learning methods for outputting structures of new compounds with stronger effects. Specifically, the research will proceed in the following three stages. 1) Development of a method for predicting drug combinations with synergistic effects by fusion analysis of omics data. 2) Network analysis of the mechanism of drug synergy. 3) Developing methods for generating and designing chemical structures of new compounds with higher synergistic effects.
  • Development of tendon/ligament injury treatment based on the understanding of its molecular mechanism
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2020/04 -2023/03 
    Author : Adachi Nobuo
     
    In this study, we focused on the functions of microRNA (miRNA) in tendons and ligaments, and investigated the effect of miRNA-containing extracellular vesicles on promoting tendon repair. Tendon- and ligament-specific Dicer knockout mice exhibited hypoplasia of tendon due to decreased expression of related genes in tendons and ligaments. Then, we identified a highly expressed miRNA in tendon tissue, and its over-introduction into tendon progenitor cells induced the expression of tendon-related genes. In Achilles tendon injury models, mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) inhibited calcification and adhesion during the tendon healing, promoted their repair. Furthermore, the glycan expressed on the surface of MSC-EVs and it was shown that it is possible to evaluate the therapeutic effect (quality) based on the glycan expression pattern.
  • Molecular mechanism of stem cell aging in the bone marrow and muscular tissues
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2020/04 -2023/03 
    Author : Teramura Takeshi
     
    Dysfunction in locomotive tissues occurs with aging is the important impediment to QOL. In this study, we investigated molecular mechanism of stem cell aging focusing on the gene regulatory mechanisms at various levels. Using bone marrow and muscular tissues from aging model mice and humans, we clarified the mechanisms causing stem cell depletion and abnormal differentiation and obtained the notion indicating the possibility of therapeutics to recover the stem cell function by gene transferring.
  • Study on the mechanisms of anti-aging food compounds by suppressing amyloid formation in gut
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2020/04 -2023/03 
    Author : Matsubara Kiminori
     
    As the formation of insoluble denatured proteins (amyloids) causes age-associated diseases, prevention of amyloid formation is important for anti-aging. Oral administration of a food-derived compound that stabilizes transthyretin (TTR), an amyloidogenic protein, in senescence-accelerated mice prone 8 (SAMP8) reduced TTR deposition, suppressed microglial activation, and maintained brain function. Amyloids also damage digest system and enhance aging. Oral administration of fermented food or microorganism using fermentation prevented changing of digest system and affected enteroendocrine cells in aged SAMP8. These results suggest that TTR-stabilizing food-derived bioactive compounds and fermented foods and microorganisms exert anti-aging effects by reducing TTR deposition and protecting the digesting system.
  • Development a novel approach for articular cartilage repair/regeneration using microneedle array sheet
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2020/04 -2023/03 
    Author : Ishikawa Masakazu
     
    This study aimed to develop a cartilage repair and regeneration therapy using microneedle array sheets based on absorbable materials and drug delivery. Although it was difficult to develop and obtain the microneedle-array-sheet, the use of human meniscus tissue demonstrated the potential of the microneedle-array-sheet to fix cartilage tissue. In addition, a novel medical device for filling and fixing articular cartilage defects with autologous tissue was explored, leading to the development of a one-step surgery for cartilage repair.
  • The role of chondrocyte/senescent cells-derived exosomal microRNA in pathogenesis of osteoarthritis, and in silico drug discoveryathogenesis of osteoarthritis and in silico drug discovery
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2022/03 
    Author : Miyaki Shigeru
     
    Osteoarthritis (OA) is the most prevalent arthritic disease. The present study focused on the role of chondrocyte/senescent cells-derived cellular/exosomal microRNA in OA pathogenesis and in silico drug discovery for OA treatment. Although miR-23 a/b cluster and miR-26a were highly expressed in articular chondrocytes, both cellular and exsomal miRNAs (miR-23a/b and miR-26a) are not essential for OA pathogenesis associated with aging, and mechanical overload and local inflammation by trauma. However, both knock out mice exhibited accelerated aging-phenotype such as bone loss. Thus, we should further examine what function of these miRNAs including exosomal miRNAs is derived from which cells, and their target genes in order to reveal the mechanism of accelerated aging-like phenotype such as osteopenia in knock out mice. These future results might open a new insight in aging mechanisms through miR-23 a/b clusters and miR-26a.
  • Development of a novel delivery system and osteosarcoma therapy using down-regulated MSC-derived exosomes
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2022/03 
    Author : Furuta Taisuke
     
    Mesenchymal stem cells (MSCs) have been reported to have the ability to migrate (homing) to tissue lesions and malignant tumors, and the development of a delivery system to add and deliver anti-tumor effectors to target cells has attracted attention. We hypothesized that exosomes, a liquid factor released from MSCs, also have a homing function and can be delivered to target cells, and verified this hypothesis using an osteosarcoma model, showing that MSC-derived exosomes may deliver miRNA143, which has antiosteosarcoma effects, to osteosarcoma cells The results also suggest that MSC-derived exosomes may have a homing function.
  • New strategy for the diagnosis of osteonecrosis of the femoral head
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2022/03 
    Author : Shoji Takeshi
     
    We evaluated disease specific microRNA in the synovial fluid of the patients with idiopathic osteonecrosis of the femoral head (ONFH) for detection of the new biomarker. We detected several microRNAs including let7c, miR-30, miR-31, miR-150, miR-210 which were highly up-regulated in ONFH patients compared with control. Furthermore, our findings revealed that the expression level of up-regulated miRNAs didn't affect the ONFH stage. We tried to detect the association specific miRNA such as steroid-related, alcohol related, but there were no specific miRNAs with statistical significance, which might be caused from small number of the ONFH patients, especially the ONFH patients without steroid and alcohol relation.
  • Systems Bone Biology
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2018/07 -2021/03 
    Author : Shinohara Masahiro
     
    Bone and cartilage tissues have central roles in daily activities, and diseases associated with a destruction of these tissues such as osteoporosis or osteoarthritis lead to deterioration of daily activity or quality of life. In this study, we established a multidiscipline research team of bone and cartilage researchers with researchers in mathematical science, and developed a basis of epoch-making systems that make prediction of disease onset and early diagnosis possible by analyzing changes over time of serum markers or tissue microstructures.
  • Therapeutic strategy for osteoarthritis by targeting neuropeptides.
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2018/04 -2021/03 
    Author : Nakasa Tomoyuki
     
    In the OA pathogenesis, cartilage degeneration due to the subchondral bone sclerosis and degenerations of the tissues such as menisci and ligament occur. Identification of factors which causes these changes will lead to develop new treatment. We focused on neuropeptides which have various functions such as bone metabolism and angiogenesis as well as pain. We showed that neuropeptides are involved in the OA pathogenesis. The expression patterns of the calcitonin gene-related peptide (CGRP)、vasoactive intestinal peptide (VIP)、substance P were examined in human OA and OA model mice. The expression of these neuropeptides increased with the sclerotic changes of the subchondral bone. Interaperitoneal administration of CGRP, VIP receptor antagonist, and substance P agonist to OA model mice could suppress the OA progression. In addition, RNA sequence of microRNA using OA model mice serum revealed some expression patterns during OA progression.
  • Development of articular cartilage repair using minced cartilage and microRNA/exosomes
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2017/04 -2020/03 
    Author : Adachi Nobuo
     
    Autologous chondrocyte implantation has been performed for large cartilage defect, but it requires two-stage surgery for chondrocyte culture. The purpose of this study was to develop a one-stage cartilage repair by atelocollagen gel embedded minced cartilage implantation. After 3 weeks culture, cell migration and proliferation in the gel and GAG contents in minced cartilage group was significantly higher than those in isolated chondrocyte group. The culture medium in the minced cartilage group contained a large amount of microRNAs which promote chondrocyte and osteoblast differentiation. For the large osteochondral defect model of rabbits, atelocollagen embedded minced cartilage implantation achieved excellent cartilage and subchondral bone repair. Atelocollagen embedded minced cartilage implantation will be good treatment option for large cartilage defect as one-step surgery.
  • Anti-aging effect of transthyretin stabilizing food-derived bioactive compounds
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2017/04 -2020/03 
    Author : Matsubara Kiminori
     
    Transthyretin (TTR) is one of the major amyloidogenic proteins. To stabilize TTR is a useful strategy to prevent age-related diseases. We screened bioactive compounds in food that stabilize TTR and prevent its amyloid formation. Several phenolic compounds and their derivatives exert TTR stabilizing effect. Two compounds were evaluated by oral administration to senescence-accelerated mice (SAM). Carnosic acid (CA) from rosemary prevented the age-associated decline of brain function. We also found that a food-derived compound prevented TTR deposition in the brain.
  • Elucidation and control of gliosis mechanism after spinal cord injury via endoplasmic reticulum stress sensor OASIS
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2017/04 -2020/03 
    Author : Kamei Naosuke
     
    The purpose of this study was to investigate the relationship between endoplasmic reticulum (ER) stress mediated by old astrocyte specifically induced substance (OASIS) and astrogliosis in spinal cord injury (SCI). We used siRNA of OASIS and mice deficient for OASIS in SCI models. In a mouse model of spinal cord contusion injury, a significant increase in OASIS mRNA on day 7 and an increase in protein on days 7 and 14 was observed in injured spinal cords. Furthermore, siRNA injection inhibited astrogliosis and hindlimb motor function recovery. On the other hand, functional recovery was better in the OASIS-deficient mice than in the wild-type mice after SCI. OASIS deletion did not inhibit astrocyte migration but reduced the excessive accumulation of N-cadherin-expressing reactive astrocytes that formed the glial scar around the injury site. In addition, OASIS deletion increased the number of serotonin-positive axons in spinal cord regions caudal to the injury site.
  • The role of microRNA in maturation of tendon /ligament
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2016/04 -2018/03 
    Author : MIYAKI Shigeru
     
    The purpose of this study is to examine the role of microRNA (miRNA) in the development and maturation of tendon/ligament using Scleraxis-Cre knock-in:Dicer flox mice. We performed miRNA expression profiling using achilles tendon tissues and Achilles tendon-derived cells. Several miRNAs highly expressed in Achilles tendon. To examine the role of miRNAs in development and maturation of tendon/ligament, we generated Scleraxis-Cre knock-in:Dicer flox mice. Dicer is one of the most important enzyme in miRNA biogenesis. Scleraxis-Cre knock-in:Dicer flox mice showed impaired tendon maturation and healing. Highly expressed miRNAs in tendon were decreased in achilles tendon of these mice. Thus, these data suggest that miRNA play an important role in maturation and healing of tendon/ligament.
  • Search for early marker of Osteoarthritis using Exosome and establish monitoring index of exercise and nutritional effects
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2016/04 -2018/03 
    Author : ISHITOBI Hiroyuki; MIYAKI Shigeru
     
    In this study, we explored the effect of carnosic acid (CA) on osteoarthritis (OA) using human synovial fibroblast and human chondrocytes. The expression of heme oxygenase-1(HO-1) was upregulated in human synovial fibroblasts and chondrocytes treated with CA in a dose-dependent manner. CA had no effect on the expression of Col2a1 in chondrocytes. CA suppressed the expression of OA-related genes in human synovial fibroblast and chondrocytes.In addition, the expression of the cartilage-degrading enzyme was suppressed. We found that CA activates miR-140 which maintains cartilage homeostasis and activates HO-1 by suppressing the expression of Bach1 gene as its molecular mechanism. These findings suggest that CA is an effective HO-1 inducer and have a potential to be a supplement for OA prevention.
  • Analysis of novel communication factor exosomes for the treatment and diagnosis of osteoarthritis
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2015/04 -2018/03 
    Author : MIYAKI Shigeru
     
    This study has focused on novel communication factor exosomes including microRNA (miRNA) in osteoarthritis (OA). We performed miRNA expression profiling in chondrocytes and exosomes using mesenchymal stem cells (MSC), normal- and OA-derived chondrocytes, and also performed glycan profiling using these samples. Several miRNAs were highly expressed in normal chondrocytes and chondrocytes-derived exosomes. These miRNAs were decreased in OA chondrocytes and OA chondrocytes-derived exosomes. Furthermore, MSC-derived exosome-formed miRNA reduced severity of OA in OA model mice. Thus, we newly generated these miRNAs knockout and cartilage specific transgenic mice. To examine the effects of chondrocytes-derived exosomes, pre-osteoclasts, pre-osteoblasts and pre-adipocytes were treated with chondrocytes-derived exosomes. Chondrocytes-derived exosomes inhibited osteoclastogenesis. These data suggest that exosomes including miRNA may play an important role in the pathogenesis and treatment of OA.
  • Acceleration of bone repair using miRNA-induced bone marrow cells for osteonecrosis
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2015/04 -2018/03 
    Author : YAMASAKI Takuma
     
    MicroRNA (miRNA) was purified from bone marrow aspirated from the patients of steroid-related osteonecrosis of the femoral head. By global analysis of miRNA, overexpression of miR-31, miR-34a, miR-146, miR-210, miR-218 were detected. In order to evaluate the potential of bone formation, each miRNA was administrated to bone differentiation-inducing medium and bone marrow-derived cells were cultured. These 5 miRNAs induced overexpression of collagen type 1A1, and miR-31, miR-146, miR-210, miR-218 induced overexpression of Runx 2. Moreover, each miRNA was administrated to pseudarthrosis in rat and was proven to accelerate bone formation by micro CT evaluation. Induction of appropriate microRNA may have a potential of acceleration of bone formation with participating autocrine effect of miRNA.
  • Preventive effect of food-derived bioactive compounds with anti-angiogenic and autophagy activities on aging.
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2014/04 -2017/03 
    Author : Matsubara Kiminori
     
    We screened autophagy activating effect of food-derived anti-angiogenic compounds and found that carnosic acid (CA) in rosemary has the effect. To evaluate the anti-aging effect of CA, CA was orally administrated to senescence-accelerated mice prone 8 (SAMP8) for a year. CA group of SAMP8 showed a lower death rate compare to control group. In histological analyses, liver and kidney of CA group were better than those of control group. In behavior tests, CA group showed better results than control group suggesting CA prevented brain function. We found that the molecular mechanism by which CA exerts anti-aging effect is attribute to activation of FoxO3a, a transcription factor relating longevity.
  • Development of a novel diagnostic system for osteochondritis dissecans of the knee using optical coherence tomography
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2014/04 -2017/03 
    Author : ISHIKAWA Masakazu
     
    Samples from atelocollagen embedded chondrocytes and mouse knee osteoarthritis (OA) model were harvested and images were obtained by optical coherence tomography (OCT) system. Obtained images and their OCT values were evaluated quantitatively by originally developed software. Using our method, OCT could demonstrate articular cartilage matrix deposition in the sample of atelocollagen with chondrocytes comparing with histological evaluation. From the animal study, it was demonstrated that femur articular cartilage in mouse OA model presented significantly lower OCT value compared to normal articular cartilage. These our data show the great potential of OCT system in orthopaedic field for quantitative evaluation of articular cartilage with noncontact manner. Moreover, the OCT probe system that enable real-time optical biopsy will be helpful to diagnose articular cartilage pathology quantitatively in clinical practice.
  • Analysis of novel communication factor exosomes for the treatment and diagnosis of osteoarthritis(Fostering Joint International Research)
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research)
    Date (from‐to) : 2016 -2017 
    Author : MIYAKI Shigeru; Lotz Martin
     
    This study has focused on novel communication factor exosomes including microRNA (miRNA) in osteoarthritis (OA). We classified OA grade and performed miRNA expression profiling in human chondrocytes and exosomes using normal- and OA-derived chondrocytes. Several miRNAs were highly expressed in normal chondrocytes and chondrocytes-derived exosomes. These miRNAs were decreased in OA chondrocytes and OA chondrocytes-derived exosomes. Thus, we newly generated cartilage-specific miRNA transgenic mice and have examined the effect of prevention by miRNA on OA. Furthermore, aging-related transcriptional factor and SOX9 regulated the miRNA and host gene, and miRNA regulated several target genes.
  • The strategy for refractory fracture using the combination of microRNA with magnetic targeting system
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2014/04 -2016/03 
    Author : OCHI MITSUO; MIYAKI SHIGERU; KAMEI NAOSUKE; NAKASA TOMOYUKI
     
    It is difficult to achieve bone union for refractory fracture cases with a non- invasive technique. MicroRNAs (miRNAs) are short, non-coding RNAs that act as repressors of gene expression at the level of post transcriptional regulation. The aim of this study was to identify miRNA which play an important role in osteogenesis, and acceleration of bone union by regulating identified miRNA in rat refractory fracture model. miR-222 was identified by microarray analysis during osteogenesis. Inhibition of miR-222 promoted osteogenesis and over-expression of miR-222 inhibited osteogenesis from human mesenchymal stem cells in vitro. miR-222 inhibitor was administered into the fracture site in rat refractory fracture model. Bone union at the fracture site was achieved in the miR-222 inhibitor administration by confirming radiographic and histological evaluation. Local administration of miR-222 inhibitor could accelerate bone healing through enhancing osteogenesis in the rat refractory model.
  • The role of exosomes as a novel communication factor in tissue regeneration
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    Date (from‐to) : 2013/04 -2016/03 
    Author : OCHI MITSUO; ADACHI NOBUO; MIYAKI SHIGERU; KAMEI NAOSUKE; NAKASA TOMOYUKI
     
    Paracrine signaling by bone-marrow-derived mesenchymal stem cells (MSCs) plays a major role in tissue repair. We focused on exosomes, which are extracellular vesicles as a novel additional modulator of cell-to-cell communication and tissue regeneration. To address this, we examined the role of exosomes in the healing process in injury models of deficient mice, a strain which is known to reduce levels and/or function of exosomes. The retardation of tissue repair in deficient mice was rescued by the injection of MSC-exosomes. MSC-exosomes promoted cell differentiation such as myogenesis and angiogenesis in vitro, and tissue regeneration in tissue injury models. The levels of the tissue repair related-cytokines in MSC-exosomes were low, suggesting that, tissue repair may be in part mediated by other MSC-exosome components, such as miRNAs. We conclude that MSC-exosomes are a novel factor of MSC paracrine signaling with an important role in the tissue repair process.
  • Development of new treatments using pro-angiogenic and tissue-specific microRNAs for the injury of musculoskeletal and nerve tissue
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2013/04 -2016/03 
    Author : KAMEI NAOSUKE; OCHI MITSUO; MIYAKI SHIGERU; NAKASA TOMOYUKI; ISHIKAWA MASAKAZU
     
    The administration of miR-210 to the mouse spinal cord injury model promoted angiogenesis and astrogliosis, and improved functional recovery after spinal cord injury compared with the non-injected controls. Additionally, the administration of miR-145 to the same model improved functional recovery and inhibited the expression of semaphorin 3A. An intra-articular injection of ds miR-210 was effective in the healing of the damaged white zone meniscus through promotion of the collagen type 2 production from meniscus cells and through upregulated of VEGF and FGF2 from synovial cells. The administration of miR-210 to the rat calcaneal tendon injury model promoted the repair of a calcaneal tendon at early phase follow injury.
  • Bach1 deficiency reduces severity of osteoarthritis, and induction of HO-1 is a novel target in OA prevention.
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2013/04 -2015/03 
    Author : MIYAKI Shigeru; NAKASA Tomoyuki; KATO Yoshio
     
    Bach1 is a transcriptional repressor of Heme oxygenase-1 (HO-1), which is cytoprotective through its antioxidant effects. The objective of this study was to define the role of HO-1 in osteoarthritis (OA) development using in Bach1 deficient mice. HO-1 expression decreased with aging in articular cartilages. Bach1 deficient mice showed reduced severity of age-related OA and surgically-induced OA compared with wild-type mice. Autophagy marker LC3 and antioxidant SOD2 were increased in articular cartilage of Bach1 deficient mice compared with wild-type mice. The expression of SOD2 and the suppression of apoptosis in Bach1 deficient chondrocytes were mediated by HO-1. This may be due to maintenance of joint health by antioxidant effects through HO-1. These results suggest that HO-1 or inactivation of Bach1 is a novel target and signal pathway in OA prevention.
  • Therapeutic strategy based on angiogenesis induce by microRNA in orthopaedic field
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2012/04 -2015/03 
    Author : NAKASA Tomoyuki; OCHI Mitsuo; MIYAKI Shigeru; KAMEI Naosuke
     
    The therapeutic effect of microRNA (miRNA) which play a role in angiogenesis on injury in orthpaedic field. Especially, we focused on secreted miRNAs from cell. Peripheral blood mononucleated cells were isolated from healthy subjects, and cultured. Stimuli by hypoxia environment was given during culture, and the medium with or without hypoxia were analyzed using miRNA microarray. Several miRNAs showed specific expression pattern, and over expression of these miRNAs in fibroblast induced cell proliferation. In vivo, administration of miRNA related to angiogenesis into rat model of meniscus, tendon injury and atrophic non-union showed successful results.
  • Exosome-formed miRNA for the treatment of osteosarcoma
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2012/04 -2015/03 
    Author : SHIMOSE Shoji; OCHI Mitsuo; KUBO Tadahiko; MIYAKI Shigeru
     
    MicroRNAs (miRNAs) have emerged as potential anticancer agents, but their clinical application is limited by the lack of an effective delivery system to tumors. Exosomes are small vesicles that play important roles in intercellular communication. Here, we show that synthetic miR-143 introduced into cells is released enveloped in exosomes and that the secreted exosome-formed miR-143 is transferred to osteosarcoma cells. The delivery of exosome-formed miR-143 significantly reduced the migration of osteosarcoma cells. The delivery efficiency of exosome-formed miR-143 was less than that achieved with lipofection, but the migratory potential of osteosarcoma cells was similarly inhibited after both strate- gies. Our results suggest that exosomes can deliver synthetic miR-143 and are a potentially efficient and functional delivery system.
  • Exosomes including microRNA as a new mediators of communication among joint cells in OA pathogenesis
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A)
    Date (from‐to) : 2012/04 -2015/03 
    Author : MIYAKI Shigeru
     
    Exosomes from IL-1β stimulated synovial fibroblasts induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints. Our observations suggest that not only established signaling molecules, such as cytokines and hormones, but also exosomes including microRNA, as mediators of communication among different joint cells and tissues play an important role in osteoarthritis pathogenesis as a new regulatory mechanism. Furthermore, exosomes including miRNAs from mesenchymal stem cells promote tissue repair from injury. These results may explain the mechanism of tissue repair, in addition to their secretion of cytokines, or growth factors and may be a new therapeutic tool.
  • cartilage regeneration using decellularized tissue, peripheral blood mononuclear cell, and microRNA
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2012/04 -2014/03 
    Author : OCHI Mitsuo; MIYAKI Shigeru; KAMEI Naosuke; NAKASA Tomoyuki
     
    The purpose of this study is to create cartilage tissue using decellularized cartilage, peripheral blood mononuclear cell and microRNA. It was impossible to induce chondrogenesis from peripheral blood mononuclear cell because the pellet culture of peripheral blood mononuclear cell could not be achieved. Osteochondral plug of rat was decellularized by 2%SDS for 12 hours, and decellularized osteochondral plug with cartilage matrix was obtained. But DNA content was not completely removed. Next, microRNA expression in the culture medium of chondrocyte sheet was analyzed. It was revealed that chondrocyte sheet secreted microRNAs which play a crucial role in chondrogenesis and osteogenesis.
  • 四肢前後軸を形成する転写ネットワークの解析
    日本学術振興会：科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2010/04 -2013/03 
    Author : 横山 成俊; 淺原 弘嗣; 五十嵐 ありさ; 味八木 茂
     
    多指症を含む四肢の形成疾患は、ヒトでは1000人に一人といった高頻度で生じ、ヒトの先天性疾患としては最も報告例の多い疾患である。現在までに多指症として221の症候群が報告されているが、原因遺伝子として記載されているのはわずかに15遺伝子のみであり、さらなる研究の進展が望まれている。 我々はヒト多指症の原因遺伝子の候補としてIrx3に注目し、マウス胚を用いて解析を行った。Irx3ノックアウトマウスを作製し、その表現型を調べたところ、後肢特異的に多指を生じることが明らかになった。ここで観察された多肢形成の様式には、親指の前側に第一指様の多肢を生じるパターンと第二指様の多肢を生じるパターンの2つが同程度の割合で混在していた。 次にこれまでに前後軸形成に関わることが知られている遺伝子についてqRT-PCR法を用いて解析を行ったところ、Irx3ノックアウトマウスにおいては、Alx4の発現量が半分程度に減少しており、またFGF4、Gremlin、dHand、Ptch1、Hoxd13、Hoxd10などの遺伝子について発現量が増加していることを明らかにした。Alx4は軸前多肢変異マウス(Strong's luxoid)の原因遺伝子であり、Alx4ヘテロマウスでは、後肢特異的に軸前多肢(第二指様)を生じることが知られている。 In situハイブリダイゼーション法を用いてこれらの遺伝子の発現領域の変化を調べたところ、Shhについては、一部の肢芽において、前側領域に異所的に発現していることを確認した。またFGF4、Gremlinについてもその発現領域が前側まで拡大していることが確認できた。 以上の研究結果を総合すると、新規転写因子Irx3は四肢前後軸形成に必須の分子ネットワークの一部を構成しており、またヒトの多指症の原因遺伝子の一つとなっている可能性を明らかにした。
  • Elucidation for mechanisms of development and regeneration in tendon and ligament
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2010 -2011 
    Author : ASAHARA Hiroshi; MIYAKI Shigeru; ITO Yoshiaki
     
    Tendon and ligament are dense, fibrous connective tissues that allow for body movement. Elucidation for mechanisms of development and regeneration in these tissues is important research subject because tendon and ligament tissues heal very slowly and rarely recovers completely. We identified Mkx as a transcription factor specifically expressing in tendon and ligament. To investigate the in vivo functions of Mkx, we generated Mkx^<-/-> mice. These mice had hypoplastic tendons throughout the body. We also observed a downregulation of type I collagen in Mkx^<-/-> tendons. These data indicate that Mkx plays a critical role in tendon differentiation. We also performed the screening for upstream and downstream factors of Mkx, and identified the candidate genes. These findings will serve as a basis for understanding molecular mechanisms of tendon differentiation and may provide a therapeutic target for tendon injuries and tendon related diseases, such as Ehlers-Danlos syndrome.
  • Non-coding RNAs regulating cartilage development
    Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2008 -2010 
    Author : ASAHARA Hiroshi; MIYAKI Shigeru; KUDO Hiroe; SATO Tempei
     
    We examined the expression pattern of miRNAs in chondrogenesis and found that miR-140 is specifically expressed in cartilage. Loss and gain of function experiments of miR-140 indicated that miR-140 is critical for both cartilage development and homeostasis, partly via regulating Adamts-5.
  • 腱の発生・再生に関わるホメオボックス遺伝子Mohawkの機能解析
    日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    Date (from‐to) : 2007 -2009 
    Author : 浅原 弘嗣; 工藤 寛枝; 味八木 茂; 浅原 弘嗣
     
    骨格を機能的につなぎ、可動させる組織として、靭帯および靭帯を構成する細胞は、生物学的にも非常に興味深い対象であり、かつ医学的にも、肩腱板、上腕骨外側上顆、アキレス腱などの炎症および断裂の治療面から見ても重要な組織であるにもかかわらず、腱や靭帯の組織形成の分子機構に関しては解明されていない。また、四肢腱形成の分子機構に焦点を当てた近年の研究から、筋、軟骨、外胚葉などと間充織との組織間相互作用が腱特異的遺伝子発現を制御することが明らかになりつつある。しかし、これまで報告されている腱関連遺伝子が腱の発生・分化、および再生機構で果たす役割は未解明の部分が多い。我々はマウス胚のin situハイブリダイゼーションによって、腱で特異的に発現し、多くの動物のボディプランに係る制御的遺伝子に共通して見られるホメオドメインをもつホメオボックス遺伝子'Mohawk'を見いだした。さらに、Mohawk欠損マウスを作製し、その表現型を解析したところ、Mohawk欠損マウスでは腱の分化が阻害されていることが分かり、また、腱の主要成分であるタイプIコラーゲンの遺伝子発現がMohawkによって制御されることを見いだした。以上よりMohawkが腱の成熟に必須の機能をもつことが明らかとなった。この成果は腱の再生・修復やエラースダンロス症候群などの病態解明に役立つことが期待される。
  • 核内蛋白質のクロマティン因子および成長因子としての内軟骨性骨化における役割の解明
    日本学術振興会：科学研究費助成事業 特定領域研究
    Date (from‐to) : 2006 -2007 
    Author : 淺原 弘嗣; 味八木 茂; 橋本 徳; 工藤 寛枝
     
    Runx2は未分化間葉系細胞の骨芽細胞への分化,骨芽細胞の増殖,I型コラーゲンなどの骨基質の産生などに必須の役割を果たす転写因子である。Runx2は分化の早い段階から石灰化にいたるまで常に発現しており,その間発現量はあまり変化しないことから,分化の各ステージにおける標的遺伝子の発現制御にはRunx2と協調して働くコファクターの存在が重要であると考えられている。本研究ではRunx2による転写を制御する転写コアクティベーターを,6xOSE2(osteoblast specific element)レポーターを用いて,ヒト全長cDNAライブラリ約10,000遺伝子からスクリーニングした。スクリーニングには,ハイスループットで細胞べースのトランスフェクションアッセイを行う系を構築した。その結果,Runx2の活性をあげる転写コファクターが同定された。さらに,その転写コファクターの骨形成における役割をノックアウトマウスによって解析したところ,ノックアウトマウスでは骨形成の遅延,阻害がみられた。これらより,Runx2の新たな制御機構が発見された。今後さらにこの遺伝子の機能を明らかにすることで,骨関節疾患の病態解明および新規治療薬の開発に役立つことが期待される.
  • 新規Znフィンガープロテインの機能解析による軟骨発生・分化のメカニズムの解明
    日本学術振興会：科学研究費助成事業 萌芽研究
    Date (from‐to) : 2005 -2007 
    Author : 浅原 弘嗣; 味八木 茂; 橋本 徳; 工藤 寛枝
     
    前年度までに我々は、前駆軟骨細胞株ATDC5を用いたDNAマイクロアレイによる解析と、マウス胚における網羅的なホールマウントin situハイブリダイゼーションにより、軟骨組織の分化に関わる転写因子、転写コファクターの同定を行ってきた。その中で、四肢、肝臓に強く発現する新規のZinc fingerモチーフを持った転写制御因子の可能性がある遺伝子を同定した。平成19年度は、これら四肢発生時に特異的に発現する新たなZinc finger遺伝子を3遺伝子(Zfp35、Zfp775、Zfp398)のうち、Zfp398遺伝子について、米国baygenomics社より、ジーントラップES細胞株を購入し、そのES細胞株を用いてマイクロインジェクション法によってキメラマウスを作製した。さらに、ジーントラップマウスを取得するべく、マウスの交配を行いジャームラインにも載っていることを確認した。本研究の目標の一つであった、ZFP遺伝子についてのジーントラップマウスを取得することに成功したことで、今後、これらの遺伝子の発現パターンをLacZ染色により確認し、さらに四肢発生を中心にフェノタイプを解析していく。本研究を継続、発展することによって、この遺伝子の発生における役割を明らかにし、骨関節疾患などの診断、治療に役立てることを期待する。
  • ヒト骨髄由来間葉系幹細胞におけるmicroRNAによる分化制御の網羅的解析
    日本学術振興会：科学研究費助成事業 特定領域研究
    Date (from‐to) : 2005 -2006 
    Author : 淺原 弘嗣; 味八木 茂; 橋本 徳; 工藤 寛枝
     
    近年、転写後発現調節にタンパク質をコードしないRNA(non-cording RNA/ncRNA)が関与することが新たに示された。miRNAの発現は、組織特異的または発生段階特異的に発現され、発生・分化を制御していると考えられる。また、幹細胞の未分化維持機構にも作用している可能性もあり、その分子機構の解明により将来的な医療応用への実現が期待される。そこで、ヒト骨髄由来間葉系幹細胞(hMSC)など未分化間葉系細胞から軟骨細胞、筋細胞などの筋骨格系細胞分化過程でその発現に変化のあるmiRNAを同定し、それらの発生・分化に対する影響を明らかにすることを目的とした。 昨年度までに、hMSCと軟骨細胞、未分化間葉系細胞であるマウスC2C12細胞において発現しているmiRNAをマイクロアレーにより網羅的な解析を行った。分化誘導にともない発現に変動のみられたmiRNAに対して、ノーザンブロット、リアルタイムPCRによりMSCで高発現するもの、各分化誘導によって発現が増加するmiRNAを同定した。同定したmiRNAは、その発現様式を調べるためにマウス胚を用いたボールマウントおよび組織In situハイブリダイゼーションを行なった。その結果、発生期において将来筋肉となる体節や心臓、軟骨形成領域に組織特異的に発現するmiRNAの詳細な発現様式を明らかにした。本年度は、これらの筋および軟骨特異的miRNAのノックアウトマウス、組織特異的トランスジェニックマウスを作製することを第一とした。今後これら遺伝子改変マウスを解析することで組織特異的miRNAの発生・分化における機能を明らかにする。以上のことより、miRNAによるMSCの分化制御機構の一端を明らかにすることで、幹細胞の未分化および分化制御の新たな基本的なメカニズムの一部を理解することに貢献する。
  • 核内蛋白HMGB1の成長因子としての骨分化におけるメカニズムの解明
    日本学術振興会：科学研究費助成事業 特定領域研究
    Date (from‐to) : 2005 -2005 
    Author : 浅原 弘嗣; 橋本 徳; 味八木 茂
     
    近年、DNAとヒストンの複合体であるクロマティンとその制御ファクターによるエピジェネティックな遺伝子発現調節が、発生を制御する重要な因子として注目されている。本研究は、クロマティン蛋白であるHMGB-1(High mobility group box protein)が、同時に肥大軟骨細胞より細胞外へ分泌される知見から、HMGB-1の未知の機能を解明し、内軟骨性骨化メカニズムと核内クロマティン蛋白質の新たな機能を明らかにすることを目的とする。これまでの我々の研究により、以下の結果が得られた。HMGB-1欠損マウス胎仔の解析より、軟骨成熟により内軟骨性骨化の始まる胎生16日において外観的に四肢の短小を認めた。その骨格形成に及ぼす影響を詳しく検討した結果、骨芽細胞への分化は認められず、内軟骨性骨化に影響を与えることを発見した。正常マウス胎仔の四肢におけるHMGB-1の免疫染色の結果、軟骨増殖層では発現が認められないが、前肥大層から肥大層にかけては、その顕著な発現を認め、その局在の多くは細胞質内であった。核内蛋白であるHMGB-1が核外細胞質内、さらには細胞外にも移行し、成長因子として作用、その血管誘導能がある可能性を見出した。正常マウス胎仔の長管骨器官培養により、その培養上清中にHMGB-1が分泌されていることを確認した。現在、in vitro系を用い、軟骨細胞内における局在の変化による影響、軟骨・骨芽細胞への分化への影響、野生型マウスとHMGB-1ノックアウトマウスの骨髄より単離した間葉系幹細胞細胞を用いて、骨芽細胞、軟骨細胞へ分化誘導することによりHMGB-1の分化能に対する影響を解析中である。我々のデータから、HMGB-1の、骨分化(内軟骨性骨化)に対する成長因子としての重要な役割が推察される。

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