Derivation of human iPS cell derived hepato-biliary organoids with bile acid secretion

• Project/Area Number: 18K08589
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 55010:General surgery and pediatric surgery-related
• Research Institution: Shinshu University
• Principal Investigator: 小川 真一郎 信州大学, 医学部, 特任教授 (30419353)
• Co-Investigator(Kenkyū-buntansha): 増田 雄一 信州大学, 学術研究院医学系(医学部附属病院), 准教授 (60467149) ; 小山 誠 信州大学, 医学部附属病院, 助教(特定雇用) (80712778)
• Project Period (FY): 2018-04-01 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2020: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2018: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: iPS細胞 / 胆管細胞 / 胆汁鬱滞 / organ on chip / 3D biliary tube / iPS cell / cholangiocyte / Primary cilia / 肝内胆管 / シングルセルRNA解析 / 肝細胞 / オルガノイド / 一次繊毛 / Mechano-sensor / Ca2+シグナル / ヒトiPS細胞 / 3次元培養 / 薬剤スクリーニング / 再生医療 / 肝臓 / 胆管 / 胆汁酸合成 / Organ on chip

Outline of Annual Research Achievements

202１年に発表したiPS細胞由来一次繊毛陽性機能的胆管細胞のシングルセルRNAシークエンス解析を行い、肝内胆管細胞特有の遺伝子プロファイルを有していることが確認できたが、ヒト肝臓サンプルを用いたシングルセルRNA解析では、胆汁酸に関連するシグナルや、fluid flowに関わるシグナルがiPS細胞由来胆管細胞に有意に減少していることが判明した。これは従来の静置培養条件化におけるオルガノイド培養の限界を表しており、この問題を解決するため、Organ on chipプラットフォームを改善し、fluid flow環境下でiPS細胞由来胆管細胞を培養することが可能な３D胆管チューブを作成した。２０２２年に発表したAngio Tubeを改良し、384ウェル培養皿上に、まずは、３つの横に連結するウェルの真ん中のウェルに３Dバイオプリンターでゲラチンチューブを作成した。このゲラチンチューブをフィブリノーゲンを主成分とするハイドロゲルで覆ったのち、PBSで洗浄することにより、ゲラチンチューブが徐々に吸収され内腔が空洞のチューブが作成することができた。このチューブは左右に存在するウェルにトンネルで繋がっており、左右のウェルに培養液を加えて、Rockerで時間経過と共に左右に傾けながら培養しることにより、（約１５度、５分インターバル）チューブ内に一定の流れを作成することができた。さらに、iPS細胞由来肝前駆細胞を播種しチューブの内腔を完全に上皮化後、胆管細胞分化誘導プロトコールを改変し、CFTR陽性、一次繊毛が内腔に突出して存在する３D胆管チューブの作成に成功した。プレート内にこの３Dチューブが１２８個作成でき、胆汁酸添加環境下における上皮接合の破綻などを胆管障害の病態モデルの作成が可能になった。これらの結果をまとめて２０２４年度内には論文に投稿予定である。

Research Products

• [Int'l Joint Research] University of Toronto/University Health Network(カナダ)
• [Int'l Joint Research] University of Toronto/University Health Network/Sick Kids Hospital Toronto(カナダ)
• [Int'l Joint Research] Oregon Health University(米国)
• [Journal Article] Viscoelastic Notch Signaling Hydrogel Induces Liver Bile Duct Organoid Growth and Morphogenesis (2022)
  • Author(s): Rizwan Muhammad、Ling Christopher、Guo Chengyu、Liu Tracy、Jiang Jia‐Xin、Bear Christine E.、Ogawa Shinichiro、Shoichet Molly S.
  • Journal Title: Advanced Healthcare Materials Volume: 11 Issue: 23 Pages: 2200880-2200880
  • DOI: 10.1002/adhm.202200880
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Subtractive manufacturing with swelling induced stochastic folding of sacrificial materials for fabricating complex perfusable tissues in multi-well plates (2022)
  • Author(s): Rajasekar Shravanthi、Lin Dawn S. Y.、Zhang Feng、Sotra Alexander、Boshart Alex、Clotet-Freixas Sergi、Liu Amy、Hirota Jeremy A.、Ogawa Shinichiro、Konvalinka Ana、Zhang Boyang
  • Journal Title: Lab on a Chip Volume: 22 Issue: 10 Pages: 1929-1942
  • DOI: 10.1039/d1lc01141c
  • Peer Reviewed / Open Access
• [Journal Article] Generation of functional ciliated cholangiocytes from human pluripotent stem cells (2021)
  • Author(s): Ogawa Mina、Jiang Jia-Xin、Xia Sunny、Yang Donghe、Ding Avrilynn、Laselva Onofrio、Hernandez Marcela、Cui Changyi、Higuchi Yuichiro、Suemizu Hiroshi、Dorrell Craig、Grompe Markus、Bear Christine E.、Ogawa Shinichiro
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 6504-6519
  • DOI: 10.1038/s41467-021-26764-0
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] One‐Step Formation of Protein‐Based Tubular Structures for Functional Devices and Tissues (2021)
  • Author(s): Gao Wuyang、Vaezzadeh Nima、Chow Kelvin、Chen Haotian、Lavender Patricia、Jeronimo Mark D.、McAllister Arianna、Laselva Onofrio、Jiang Jia‐Xin、Gage Blair K.、Ogawa Shinichiro、Ramchandran Arun、Bear Christine E.、Keller Gordon M.、G?nther Axel
  • Journal Title: Advanced Healthcare Materials Volume: 10 Issue: 8 Pages: 2001746-2001746
  • DOI: 10.1002/adhm.202001746
• [Journal Article] Building consensus on definition and nomenclature of hepatic, pancreatic, and biliary organoids. (2021)
  • Author(s): Marsee A, Roos FJM, Verstegen MMA; HPB Organoid Consortium*, Gehart H, de Koning E, Lemaigre F, Forbes SJ, Peng WC, Huch M, Takebe T, Vallier L, Clevers H, van der Laan LJW, Spee B.（*Hepatic, Pancreatic, and Biliary (HPB) Organoid Consortium のメンバーとして参加）
  • Journal Title: Cell Stem Cell Volume: 28 Issue: 5 Pages: 816-832
  • DOI: 10.1016/j.stem.2021.04.005
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Generation of functional ciliated cholangiocytes from human pluripotent stem cells (2021)
  • Author(s): Ogawa Mina、Jiang Jia-Xin、Xia Sunny、Yang Donghe、Ding Avrilynn、Laselva Onofrio、Chin Stephanie、Hernandez Marcela、Cui Changyi、Higuchi Yuichiro、Suemizu Hiroshi、Dorrell Craig、Grompe Markus、Bear Christine E、Keller Gordon、Ogawa Shinichiro
  • Journal Title: bioRxiv Volume: -
  • DOI: 10.1101/2021.03.23.436530
  • Open Access / Int'l Joint Research
• [Journal Article] Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations (2018)
  • Author(s): MacParland Sonya A.、Liu Jeff C. Ogawa S, Keller G, Bader G, McGilvray I
  • Journal Title: Nature Communications Volume: 9 Issue: 1 Pages: 1-21
  • DOI: 10.1038/s41467-018-06318-7
  • Peer Reviewed / Open Access
• [Presentation] Developing Targeted Cell Therapies for liver disease from Human Pluripotent Stem cells (2023)
  • Author(s): Shinichiro Ogawa
  • Organizer: Till & McCulloch meeting 2023, Toronto, October 23-25
  • Int'l Joint Research / Invited
• [Presentation] Generation of functional ciliated cholangiocytes from human pluripotent stem cells (2022)
  • Author(s): Mina Ogawa, Jia-Xin Jiang, Sunny Xia, Donghe Yang, Avrilynn Ding, Onofrio Laselva, Marcela Hernandez, Changyi Cui, Christine E Bear, and Shinichiro Ogawa
  • Organizer: 2022/10/03 Till and McCullogh Meeting
  • Int'l Joint Research
• [Presentation] hPSC-derived biliary epithelial cells. functional role and future applications (2022)
  • Author(s): Mina Ogawa, Shinichiro Ogawa
  • Organizer: University of Toronto, Medicine by design, workshop
  • Int'l Joint Research / Invited
• [Presentation] How do Other Organs Vary (2020)
  • Author(s): Mina Ogawa, Jia-Xin Jiang, Sunny Xia, Christine E. Bear, and Shinichiro Ogawa
  • Organizer: North American Cystic Fibrosis Conference 2020 Virtual
  • Int'l Joint Research / Invited
• [Presentation] The efficient production of functional cholangiocyte from human pluripotent stem cells (2020)
  • Author(s): Mina Ogawa, Yuichiro Higuchi, Hiroshi Suemizu, Donghe Yang, Gordon Keller, and Shinichiro Ogawa
  • Organizer: The 19th Congress of the Japanese Society for Regenerative Medicine Virtual
• [Presentation] The efficient production of functional cholangiocytes from human pluripotent stem cells (2019)
  • Author(s): Mina Ogawa, Shinichiro Ogawa
  • Organizer: International Society for stem cells Research (ISSCR) Annual Meeting
  • Int'l Joint Research
• [Presentation] Drug screening for CFTR modulators in iPSCs derived cholangiocytes from Cystic Fibrosis patients (2019)
  • Author(s): Mina Ogawa, Shinichiro Ogawa
  • Organizer: Till and McCulloch meeting 2019, Canada
  • Int'l Joint Research