ヒト骨発生機構の理解に基づく組織再生研究基盤の構築

• Project/Area Number: 21H04952
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Medium-sized Section 90:Biomedical engineering and related fields
• Research Institution: The University of Tokyo
• Principal Investigator: 鄭 雄一 東京大学, 大学院工学系研究科(工学部), 教授 (30345053)
• Co-Investigator(Kenkyū-buntansha): 大庭 伸介 大阪大学, 大学院歯学研究科, 教授 (20466733) ; 酒井 崇匡 東京大学, 大学院工学系研究科(工学部), 教授 (70456151) ; 北條 宏徳 東京大学, 大学院医学系研究科(医学部), 准教授 (80788422)
• Project Period (FY): 2021-04-05 – 2025-03-31
• Project Status: Granted (Fiscal Year 2024)
• Budget Amount: ¥40,950,000 (Direct Cost: ¥31,500,000、Indirect Cost: ¥9,450,000); Fiscal Year 2024: ¥8,840,000 (Direct Cost: ¥6,800,000、Indirect Cost: ¥2,040,000); Fiscal Year 2023: ¥10,660,000 (Direct Cost: ¥8,200,000、Indirect Cost: ¥2,460,000); Fiscal Year 2022: ¥10,140,000 (Direct Cost: ¥7,800,000、Indirect Cost: ¥2,340,000); Fiscal Year 2021: ¥11,310,000 (Direct Cost: ¥8,700,000、Indirect Cost: ¥2,610,000)
• Keywords: 骨再生 / 幹細胞 / １細胞解析 / バイオマテリアル / 一細胞解析 / hPSCs / 骨誘導 / ヒト多能性幹細胞 / ゲノム編集

Outline of Research at the Start

これまで再生医療の基礎研究では、主に齧歯類がモデル動物として用いられてきましたが、ヒトと齧歯類の間で骨再生性シグナルに対する反応性が異なることが分かってきました。そこで本研究では、当研究室が確立したヒト多能性幹細胞の三次元骨組織誘導法を用いて、ヒト骨発生機構を解明します。ヒトとマウスの骨発生機構の比較解析をすることで、ヒト骨発生に重要なシグナル因子を明らかにします。さらに、得られたシグナル因子をバイオマテリアルと統合することで、骨再生性足場ユニットを創製し、骨再生効果を実証します。これによりヒト骨発生機構の理解に基づく新たな骨組織再生法の確立を目指します。

Outline of Annual Research Achievements

本研究では、ヒト骨発生機構の理解に基づく骨再生研究基盤の構築を目指し、以下の４つのプロジェクトを遂行する：1.ヒト骨発生機構の解析、2.ヒト骨発生制御シグナル因子群の抽出、3.シグナル因子群の機能・階層性の検証、4.ヒト骨再生性シグナルと足場材料の統合と骨欠損モデルを用いたPOC検証。
本年度は昨年度に引き続き上記1,2のプロジェクトを進めた。当研究室で開発したヒト多能性幹細胞由来三次元骨組織を用いた遺伝子制御ネットワークの解析を行った。ヒト多能性幹細胞から椎板細胞（骨軟骨前駆細胞）へのStepwise分化誘導と、誘導細胞の免疫不全マウス腎被膜下への移植により三次元骨組織を誘導する。誘導した骨組織から細胞を回収した後、細胞核を抽出し、一細胞マルチオーム解析（Single Cell RNA-seq＋Single Cell ATAC-seq）を行った。本解析では、クロマチン状態と遺伝子発現を一細胞単位で同時に検出することが可能である。クロマチンが開いている状態は、遺伝子発現を正に制御するエンハンサー活性と高い相関があることから、ヒト骨発生におけるダイナミックなエンハンサー活性と遺伝子発現機構の解明に取り組んだ。マルチモーダルなクラスタリング解析により、骨組織に存在する骨格前駆細胞、軟骨・肥大軟骨細胞、骨芽細胞に相当する10の細胞集団の特徴を明らかにした。得られた細胞種特異的なシグナルー転写因子群を同定するため、オープンクロマチン領域におけるモチーフ活性と遺伝子発現を統合解析し、有意水準以上の転写因子群と関連するシグナル因子を抽出した。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

予定通り有望因子の絞り込みが進んでいる。これまでの研究成果も発表できた。

Strategy for Future Research Activity

計画通り有望因子の機能解析実験を中心に研究を進める。

Research Products

• [Int'l Joint Research] 南カリフォルニア大学/コネティカット大学(米国)
• [Journal Article] Stem cell-based modeling and single-cell multiomics reveal gene-regulatory mechanisms underlying human skeletal development (2023)
  • Author(s): Tani Shoichiro, Okada Hiroyuki, Onodera Shoko, Chijimatsu Ryota, Seki Masahide, Suzuki Yutaka, Xin Xiaonan, Rowe David W., Saito Taku, Tanaka Sakae, Chung Ung-il, Ohba Shinsuke, Hojo Hironori
  • Journal Title: Cell Reports Volume: 42 Issue: 4 Pages: 112276-112276
  • DOI: 10.1016/j.celrep.2023.112276
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Single-cell RNA sequencing unravels heterogeneity of skeletal progenitors and cell-cell interactions underlying the bone repair process (2022)
  • Author(s): Nakayama Mika、Okada Hiroyuki、Seki Masahide、Suzuki Yutaka、Chung Ung-il、Ohba Shinsuke、Hojo Hironori
  • Journal Title: Regenerative Therapy Volume: 21 Pages: 9-18
  • DOI: 10.1016/j.reth.2022.05.001
  • Peer Reviewed / Open Access
• [Journal Article] Sp7 Action in the Skeleton: Its Mode of Action, Functions, and Relevance to Skeletal Diseases (2022)
  • Author(s): Hojo Hironori、Ohba Shinsuke
  • Journal Title: International Journal of Molecular Sciences Volume: 23 Issue: 10 Pages: 5647-5647
  • DOI: 10.3390/ijms23105647
  • Peer Reviewed / Open Access
• [Journal Article] Runx2 regulates chromatin accessibility to direct the osteoblast program at neonatal stages (2022)
  • Author(s): Hojo Hironori, Saito Taku, He Xinjun, Guo Qiuyu, Onodera Shoko, Azuma Toshifumi, Koebis Michinori, Nakao Kazuki, Aiba Atsu, Seki Masahide, Suzuki Yutaka, Okada Hiroyuki, Tanaka Sakae, Chung Ung-il, McMahon Andrew P., Ohba Shinsuke
  • Journal Title: Cell Reports Volume: 40 Issue: 10 Pages: 111315-111315
  • DOI: 10.1016/j.celrep.2022.111315
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Orally administrable peptides derived from egg yolk promote skeletal repair and ameliorate degenerative skeletal disorders in mouse models (2022)
  • Author(s): Kitaura Yoshiaki、Nakamura Utano、Awada Chihiro、Yamaguchi Motonori、Kim Mujo、Ikeda Yuki、Matsuo Yuki、Moriishi Takeshi、Sawase Takashi、Chung Ung-il、Hojo Hironori、Ohba Shinsuke
  • Journal Title: Regenerative Therapy Volume: 21 Pages: 584-595
  • DOI: 10.1016/j.reth.2022.11.002
  • Peer Reviewed / Open Access
• [Journal Article] Involvement of activator protein-1 family members in β-catenin and p300 association on the genome of PANC-1 cells (2022)
  • Author(s): Doi Tomomitsu、Hojo Hironori、Ohba Shinsuke、Obayashi Kunie、Endo Motoyoshi、Ishizaki Toshimasa、Katoh Akira、Kouji Hiroyuki
  • Journal Title: Heliyon Volume: 8 Issue: 2 Pages: e08890-e08890
  • DOI: 10.1016/j.heliyon.2022.e08890
  • Peer Reviewed / Open Access
• [Journal Article] A neomorphic variant in SP7 alters sequence specificity and causes a high-turnover bone disorder (2022)
  • Author(s): Lui Julian C.、Raimann Adalbert、Hojo Hironori、Dong Lijin、Roschger Paul、Kikani Bijal、Wintergerst Uwe、Fratzl-Zelman Nadja、Jee Youn Hee、Haeusler Gabriele、Baron Jeffrey
  • Journal Title: Nature Communications Volume: 13 Issue: 1 Pages: 700-700
  • DOI: 10.1038/s41467-022-28318-4
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Star-Polymer-DNA Gels Showing Highly Predictable and Tunable Mechanical Responses (2022)
  • Author(s): Ohira Masashi、Katashima Takuya、Naito Mitsuru、Aoki Daisuke、Yoshikawa Yusuke、Iwase Hiroki、Takata Shin‐ichi、Miyata Kanjiro、Chung Ung‐il、Sakai Takamasa、Shibayama Mitsuhiro、Li Xiang
  • Journal Title: Advanced Materials Volume: 34 Issue: 13 Pages: 2108818-2108818
  • DOI: 10.1002/adma.202108818
  • Peer Reviewed
• [Journal Article] Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin (2021)
  • Author(s): Wang JS, Kamath T, Mazur CM, Mirzamohammadi F, Rotter D, Hojo H, Castro CD, Tokavanich N, Patel R, Govea N, Enishi T, Wu Y, da Silva Martins J, Bruce M, Brooks DJ, Bouxsein ML, Tokarz D, Lin CP, Abdul A, Macosko EZ, Fiscaletti M, Munns CF, Ryder P, Kost-Alimova M, Byrne P, Cimini B, Fujiwara M, Kronenberg HM, Wein MN.
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 6271-6271
  • DOI: 10.1038/s41467-021-26571-7
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Induction and expansion of human PRRX1+ limb-bud-like mesenchymal cells from pluripotent stem cells (2021)
  • Author(s): Yamada Daisuke、Nakamura Masahiro、Takao Tomoka、Takihira Shota、Yoshida Aki、Kawai Shunsuke、Miura Akihiro、Ming Lu、Yoshitomi Hiroyuki、Gozu Mai、Okamoto Kumi、Hojo Hironori、Kusaka Naoyuki、Iwai Ryosuke、Nakata Eiji、Ozaki Toshifumi、Toguchida Junya、Takarada Takeshi
  • Journal Title: Nature Biomedical Engineering Volume: 5 Issue: 8 Pages: 926-940
  • DOI: 10.1038/s41551-021-00778-x
  • Peer Reviewed
• [Journal Article] Mesenchymal stromal cells in the bone marrow niche consist of multi-populations with distinct transcriptional and epigenetic properties (2021)
  • Author(s): Kanazawa Sanshiro、Okada Hiroyuki、Hojo Hironori、Ohba Shinsuke、Iwata Junichi、Komura Makoto、Hikita Atsuhiko、Hoshi Kazuto
  • Journal Title: Scientific Reports Volume: 11 Issue: 1 Pages: 15811-15811
  • DOI: 10.1038/s41598-021-94186-5
  • Peer Reviewed / Open Access
• [Journal Article] Temperature Dependence of Polymer Network Diffusion (2021)
  • Author(s): Takeshi Fujiyabu, Takamasa Sakai, Ryota Kudo, Yuki Yoshikawa, Takuya Katashima, Ung-il Chung, Naoyuki Sakumichi
  • Journal Title: Physical Review Letters Volume: 127 Issue: 23 Pages: 237801-237801
  • DOI: 10.1103/physrevlett.127.237801
  • Peer Reviewed
• [Journal Article] On-demand retrieval of cells three-dimensionally seeded in injectable thioester-based hydrogels (2021)
  • Author(s): Ishikawa Shohei、Kamata Hiroyuki、Chung Ung-il、Sakai Takamasa
  • Journal Title: RSC Advances Volume: 11 Issue: 38 Pages: 23637-23643
  • DOI: 10.1039/d1ra01934a
  • Peer Reviewed
• [Presentation] Elucidating gene regulatory mechanisms underlying human skeletal development using human pluripotent stem cell-derived bone tissuesand single-cell multiome analysis (2022)
  • Author(s): Shoichiro Tani, Hiroyuki Okada, Masahide Seki, Yutaka Suzuki, Taku Saito, Sakae Tanaka, Ung-il Chung, Hironori Hojo and Shinsuke Ohba
  • Organizer: ECTS 2022
  • Int'l Joint Research
• [Presentation] ELUCIDATING GENE REGULATORY NETWORKS UNDERLYING HUMAN SKELETAL DEVELOPMENT BY INTEGRATING SINGLE-CELL DYNAMICS OF CHROMATIN ACCESSIBILITY AND GENE EXPRESSION (2022)
  • Author(s): Shoichiro Tani, Hiroyuki Okada, Masahide Seki, Yutaka Suzuki, Ung-il Chung, Hironori Hojo and Shinsuke Ohba
  • Organizer: ISSCR 2022
  • Int'l Joint Research
• [Presentation] Elucidating Gene Regulatory Networks Underlying Human Skeletal Development by Integrating a Model of Human Endochondral Bones and Single-cell Multiomics (2022)
  • Author(s): Shoichiro Tani, Hiroyuki Okada, Masahide Seki, Yutaka Suzuki, Taku Saito, Sakae Tanaka, Ung-il Chung, Hironori Hojo and Shinsuke Ohba
  • Organizer: ASBMR 2022
  • Int'l Joint Research
• [Presentation] EXPLORING HUMAN SKELETAL DEVELOPMENT USING AN INDUCED HUMAN BONE TISSUE DERIVED FROM HUMAN PLURIPOTENT STEM CELLS (2021)
  • Author(s): Shoichiro Tani, Hiroyuki Okada, Masahide Seki, Yutaka Suzuki, Ung-il Chung, Hironori Hojo and Shinsuke Ohba
  • Organizer: ISSCR 2021
  • Int'l Joint Research
• [Presentation] Understanding a Mechanism Underlying Bone Repair Process by Combinational Analysis of Lineage Tracing and Single-Cell RNA Sequencing (2021)
  • Author(s): Mika Nakayama, Hiroyuki Okada, Masahide Seki, Yutaka Suzuki, Ung-il Chung, Shinsuke Ohba, Hironori Hojo
  • Organizer: ISSCR 2021
  • Int'l Joint Research
• [Presentation] "ELUCIDATING HUMAN SKELETAL DEVELOPMENT BY INTEGRATING SINGLE-CELL ANALYSIS AND BONE TISSUE INDUCTION USING HUMAN PLURIPOTENT STEM CELLS" (2021)
  • Author(s): Shoichiro Tani, Hiroyuki Okada, Masahide Seki, Yutaka Suzuki, Taku Saito, Sakae Tanaka, Ung-il Chung, Hironori Hojo and Shinsuke Ohba
  • Organizer: ISSCR tokyo
• [Presentation] ヒト多能性幹細胞を用いた骨形成過程の再現と応用 (2021)
  • Author(s): "谷 彰一郎, 岡田 寛之, 関 真秀, 鈴木 穣, 齊藤 琢, 田中 栄, 鄭 雄一, 北條 宏徳, 大庭 伸介 "
  • Organizer: 第36回日本整形外科学会基礎学術集会
• [Presentation] ヒト多能性幹細胞由来3次元骨組織誘導法と単一細胞解析を用いたヒト骨形成機構の探索 (2021)
  • Author(s): 谷 彰一郎, 岡田 寛之, 齊藤 琢, 田中 栄, 鄭 雄一, 北條 宏徳, 大庭 伸介
  • Organizer: 第39回日本骨代謝学会