Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus

2016 Fiscal Year Annual Research Report

• Project/Area Number: 15K15285
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: 朝比奈 靖浩 東京医科歯科大学, 大学院医歯学総合研究科, 寄附講座教授 (00422692)
• Co-Investigator(Kenkyū-buntansha): 柿沼 晴 東京医科歯科大学, 大学院医歯学総合研究科, 寄附講座講師 (30372444)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Keywords: iPS細胞 / B型肝炎ウイルス / cccDNA / NTCP

Outline of Annual Research Achievements

HBVは成熟肝細胞でのみに感染するとされるが、成熟肝細胞が一定の速度でturn overしているにも関わらず、cccDNAが核内に残存し続ける原因は不明である。本研究では、より未分化な形質を持つ細胞においてもcccDNAが保存されるかを解明するとともに、新規治療の創出に資する新たな知的・技術的基盤を確立することを目的とした。より高感度かつ簡便に検出可能な蛍光色素組み換え体HBVを用いて、未分化な形質を持つヒトiPS細胞由来肝幹・前駆細胞(iPS-HPC)において検討を行ったところ、HBV感染とcccDNA産生を確認し得た。一方、成熟化肝細胞株(iPS-Hep)ではNTCPはiPS-HPCと比較して高発現しており、HBV感染効率も高かった。この様に細胞分化度の違いによるHBV感染・増殖の差が明らかとなったが、iPS-Hepでは成熟化により長期維持培養が困難であり、長期間のウイルス動態を観察することは困難であった。そこで、感染性は比較的低いが長期培養が可能であるiPS-HPCsにおいて、NTCPを誘導性に強制発現できるiPS-HPC-NTCP株を樹立した。このiPS-HPC-NTCPではDox容量依存的にNTCPの発現増加がみられ、親株より有意にHBV感染性を認めた。さらに異なる機序の抗HBV薬効果も評価可能であった。iPS-HPC-NTCPは生理的な自然免疫応答が維持されており、JAKiによってIFN応答経路を抑制した群ではcccDNAの発現が有意に高く、cccDNAの長期維持には自然免疫応用の制御が関与していることが示唆されたiPS-HPC-NTCPおよびiPS-Hep細胞は細胞分化を基軸とした治療標的の探索および有用な創薬開発モデルとなり得ると考えられた。

Research Products

• [Journal Article] Hepatitis B Surface Antigen Reduction by Switching from Long-term Nucleoside/nucleotide Analog Administration to Pegylated Interferon. (2017)
  • Author(s): Tamaki N, Kurosaki M, Kusakabe A, Orito E, Joko K, Kojima Y, Kimura H, Uchida Y, Hasebe C, Asahina Y, Izumi N.
  • Journal Title: J Viral Hepat Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1111/jvh.12691
  • Peer Reviewed
• [Journal Article] Genome-wide association study identifies 1 a TLL1 variant associated with development of hepatocellular carcinoma after eradication of hepatitis C virus (2017)
  • Author(s): Matsuura K, Sawai H, Ikeo K, Ogawa S, Iio E, Isogawa M, Shimada N, Komori A, Toyoda H, Kumada T, Namisaki T, Yoshiji H, Sakamoto N, Nakagawa M, Asahina Y, et al.
  • Journal Title: Gastroenterology Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1053/j.gastro.2017.01.041
  • Peer Reviewed
• [Journal Article] ITPA gene variation and ribavirin-induced anemia in patients with genotype 2 chronic hepatitis C treated with sofosbuvir plus ribavirin. (2017)
  • Author(s): Murakawa M, Asahina Y, Nagata H, Nakagawa M, Kakinuma S, Nitta S, Kawai-Kitahata F, Otani S, Kaneko S, Miyoshi M, Tsunoda T, Asano Y, Sato A, Itsui Y, Azuma S, Nouchi T, Furumoto Y, Asano T, Chuganji Y, Tohda S, Watanabe M.
  • Journal Title: Hepatol Res Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1111/hepr.12867
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Hepatic IFNL4 expression is associated with non-response to interferon-based therapy through the regulation of basal interferon-stimulated gene expression in chronic hepatitis C patients. (2017)
  • Author(s): Murakawa M, Asahina Y, Kawai-Kitahata F, Nakagawa M, Nitta S, Otani S, Nagata H, Kaneko S, Asano A, Tsunoda T, Miyoshi M, Itsui Y, Azuma S, Kakinuma S, Tanaka Y, Iijima S, Tsuchiya K, Izumi N, Tohda T, and WatanabeM.
  • Journal Title: J Med Virol Volume: 89 Pages: 1241-1247
  • DOI: 10.1002/jmv.24763
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Complex pattern of resistance-associated substitutions of hepatitis C virus after daclatasvir/asunaprevir treatment failure. (2016)
  • Author(s): Itakura J, Kurosaki M, Hasebe C, Osaki Y, Joko K, Yagisawa H, Sakita S, Okushin H, Satou T, Hisai H, Abe T, Tsuji K, Tamada T, Kobashi H, Mitsuda A, Ide Y, Ogawa C, Tsuruta S, Takaguchi K, Murakawa M, Asahina Y, Enomoto N, Izumi N.
  • Journal Title: PLoS One Volume: － Pages: - e0165339
  • DOI: 10.1371/journal.pone.0165339
  • Peer Reviewed / Open Access
• [Journal Article] Effects of Resistance-Associated NS5A Mutations in Hepatitis C Virus on Viral Production and Susceptibility to Antiviral Reagents. (2016)
  • Author(s): Nitta S, Asahina Y, Matsuda M, Yamada N, Sugiyama R, Masaki T, Suzuki R, Kato N, Watanabe M, Wakita T, Kato T.
  • Journal Title: Sci Rep Volume: 6 Pages: - 34652
  • DOI: 10.1038/srep34652
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Bone Morphogenetic Protein-4 Modulates Proliferation and Terminal Differentiation of Fetal Hepatic Stem/Progenitor Cells. (2016)
  • Author(s): Goto F, Kakinuma S, Miyoshi M, Tsunoda T, Kaneko S, Sato A, Asano Y, Otani S, Azuma S, Nagata H, Kawai-Kitahata F, Murakawa M, Nitta S, Itsui Y, Nakagawa M, Asahina Y, Watanabe M.
  • Journal Title: Hepatol Res Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1111/hepr.12823
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Irregular vascular pattern by contrast-enhanced ultrasonography and high serum Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein level predict poor outcome after successful radiofrequency ablation in patients with early-stage hepatocellular carcinoma. (2016)
  • Author(s): Takada H, Tsuchiya K, Yasui Y, Nakakuki N, Tamaki N, Nakanishi H, Itakura J, Takahashi Y, Kurosaki M, Asahina Y, Enomoto N, Izumi N.
  • Journal Title: Cancer Med Volume: 5 Pages: 3111-3120
  • DOI: 10.1002/cam4.932
  • Peer Reviewed
• [Journal Article] Serial measurement of Wisteria floribunda agglutinin positive Mac-2-binding protein is useful for predicting liver fibrosis and the development of hepatocellular carcinoma in chronic hepatitis C patients treated with IFN-based and IFN-free therapy. (2016)
  • Author(s): Nagata H, Nakagawa M, Nishimura-Sakurai Y, Asano Y, Tsunoda T, Miyoshi M, Kaneko S, Goto F, Otani S, Kawai-Kitahata F, Murakawa M, Nitta S, Itsui Y, Seishin Azuma S, Kakinuma S, Tojo N, Tohda T, Asahina Y, Watanabe M, the Ochanomizu Liver Conference Study Group.
  • Journal Title: Hepatol Int Volume: 10 Pages: 956-964
  • DOI: 10.1007/s12072-016-9754-1
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Human induced pluripotent stem cell-derived hepatic cell line as a new model for host interaction with hepatitis B virus. (2016)
  • Author(s): Kaneko S, Kakinuma S, Asahina Y, Kamiya A, Miyoshi M, Tsunoda T, Nitta S, Asano Y, Nagata H, Otani S, Kitahata F, Murakawa M, Itsui Y, Nakagawa M, Azuma S, Nakauchi H, Iwamoto M, Watashi K, Wakita T, Sugiyama M, Mizokami M, Shimotohno K, Watanabe M.
  • Journal Title: Sci Rep Volume: 6 Pages: - 29358
  • DOI: 10.1038/srep29358
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Evaluation of Interferon Resistance in Newly Established Genotype 1b Hepatitis C Virus Cell Culture System. (2016)
  • Author(s): Taniguchi M, Tasaka-Fujita M, Nakagawa M, Watanabe T, Kawai-Kitahata F, Otani S, Goto F, Nagata H, Kaneko S, Nitta S, Murakawa M, Nishimura-Sakurai Y, Azuma S, Itsui Y, Mori K, Yagi S, Kakinuma S, Asahina Y, Watanabe M.
  • Journal Title: J Clin Transl Hepatol Volume: 4 Pages: 5-11
  • DOI: 10.14218/JCTH.2015.00047
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Matrix Metalloproteinase-14 Mediates Formation of Bile Ducts and Hepatic Maturation of Fetal Hepatic Progenitor Cells. (2016)
  • Author(s): Otani S, Kakinuma S, Kamiya A, Goto F, Kaneko S, Miyoshi M, Tsunoda T, Asano Y, Kawai-Kitahata F, Nitta S, Nakata T, Okamoto O, Itsui Y, Nakagawa M, Azuma S, Asahina Y, Yamaguchi T, Koshikawa N, Seiki M, Nakauchi H, Watanabe M.
  • Journal Title: Biochem Biophys Res Commun Volume: 469 Pages: 1062-1068
  • DOI: 10.1016/j.bbrc.2015.12.105
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Comprehensive analyses of mutations and hepatitis B virus integration in hepatocellular carcinoma with clinicopathological features. (2016)
  • Author(s): Kawai-Kitahata F*, Asahina Y*, Tanaka S, Kakinuma S, Murakawa M, Nitta S, Watanabe T, Otani S, Taniguchi M, Goto F, Nagata H, Kaneko S, Tasaka-Fujita M, Nishimura-Sakurai Y, Azuma S, Itsui Y, Nakagawa M, Tanabe M , Takano S, Fukasawa M, Sakamoto M, Maekawa S, Enomoto N, Watanabe M.
  • Journal Title: J Gastroenterol Volume: 51 Pages: 473-486
  • DOI: 10.1007/s00535-015-1126-4
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Efficacy of additional radiofrequency ablation after transcatheter arterial chemoembolization for intermediate hepatocellular carcinoma. (2016)
  • Author(s): Azuma S, Asahina Y, Nishimura-Sakurai Y, Kakinuma S, Kaneko S, Nagata H, Goto F, Ootani S, Kawai-Kitahata F, Taniguchi M, Murakawa M, Watanabe T, Tasaka-Fujita M, Itsui Y, Nakagawa M, Watanabe M.
  • Journal Title: Hepatol Res Volume: 46 Pages: 312-319
  • DOI: 10.1111/hepr.12566
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] JSH Guidelines for the Management of Hepatitis C Virus Infection: A 2016 update for genotype 1 and 2. (2016)
  • Author(s): Asahina Y, Izumi N, Hiromitsu K, Kurosaki M, Koike K, Suzuki F, Takikawa H, Tanaka A, Tanaka E, Tanaka Y, Tsubouchi H, Hayashi N, Hiramatsu N, Yotsuyanagi H.
  • Journal Title: Hepatol Res Volume: 46 Pages: 129-165
  • DOI: 10.1111/hepr.12645
  • Peer Reviewed
• [Journal Article] Need for appropriate programs for prevention of hepatitis B infection: lessons from the latest nationwide survey in Japan. (2016)
  • Author(s): Asahina Y and Watanabe M.
  • Journal Title: J Gastroenterol Hepatol Volume: 31 Pages: 12-13
  • DOI: 10.1111/jgh.13157
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] Reduced changes in bone mineral density in CHB patients receiving tenofovir alfenamide (TAF) compared with tenofovir disoproxil fumarate. (2016)
  • Author(s): Seto WK, Asahina Y, Peng, Stanciu CYC, Abdurakhmanov D, Flaherty JF, Kim K, Gaggar A, Subramanian M, Tabak F, Tuan T. Nguyen TT, Chan HLT
  • Organizer: The 67th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2016).
  • Place of Presentation: Boston, USA
  • Year and Date: 2016-11-11 – 2016-11-15
  • Int'l Joint Research / Invited
• [Presentation] Genetically modified human induced pluripotent stem cell-derived hepatic progenitor-like cell lines as a model for interaction between hepatitis В virus and host cells. (2016)
  • Author(s): Kaneko S, Kakinuma S, Asahina Y, Kamiya A, Miyoshi M, Tsunoda T, Nitta S, Sato A, Asano Y, Nagata H, Otani S, Kawai-Kitahata F, Murakawa M, Nakagawa M, Azuma S, Watanabe M.
  • Organizer: The 67th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2016).
  • Place of Presentation: Boston, USA
  • Year and Date: 2016-11-11 – 2016-11-15
  • Invited
• [Presentation] B型肝炎治療のアップデート HBV排除にむけたヒトiPS細胞由来肝細胞系譜細胞を用いた培養系の開発． (2016)
  • Author(s): 金子 俊, 柿沼 晴, 朝比奈 靖浩
  • Organizer: 第20回日本肝臓学会大会
  • Place of Presentation: 神戸コンベンションセンター（兵庫県神戸市）
  • Year and Date: 2016-11-03 – 2016-11-06
• [Presentation] Recent progress in anti-HCV therapy and challenges toward HCC eradication. (2016)
  • Author(s): Asahina Y.
  • Organizer: Academic Forum of Liver Disease in Northeast China and the Annual Convention for the Study of Liver Diseases in Jilin Province in 2016
  • Place of Presentation: Changchun, China
  • Year and Date: 2016-10-29 – 2016-11-20
  • Int'l Joint Research / Invited
• [Presentation] Hepatocarcinogenesis after hepatitis C viral eradication (2016)
  • Author(s): Asahina Y
  • Organizer: The 10th APASL Single Topic Conference
  • Place of Presentation: Kaoshiung, Taiwan
  • Year and Date: 2016-06-08 – 2016-06-08
  • Int'l Joint Research / Invited
• [Presentation] B型肝炎研究のUp-To-Date ヒトiPS細胞由来肝細胞系譜細胞を用いた新たなHBV感染培養系モデル (2016)
  • Author(s): 金子 俊, 柿沼 晴, 朝比奈 靖浩
  • Organizer: 第52回日本肝臓学会総会
  • Place of Presentation: ホテルニューオータニ幕張、東京ベイ幕張ホール（千葉県美浜区）
  • Year and Date: 2016-05-19 – 2016-05-20
• [Presentation] B型肝炎ウイルス排除にむけて 創薬への新知見 ヒトiPS細胞由来肝細胞系譜細胞を用いた創薬を目指したHBV感染培養研究 (2016)
  • Author(s): 金子 俊, 朝比奈 靖浩, 渡辺 守
  • Organizer: 第102回日本消化器病学会総会
  • Place of Presentation: 京王プラザホテル（東京都新宿区）
  • Year and Date: 2016-04-21 – 2016-04-23
• [Presentation] A model for interaction between HBV and host cells derived from human induced pluripotent stem cells. (2016)
  • Author(s): Kaneko S, Kakinuma S, Asahina Y, Kamiya A, Nitta S, Tsunoda T, Miyoshi M, Asano Y, Nagata H, Goto F, Otani S, Kitahata F, Murakawa M, Itsui Y, Nakagawa M, Azuma AS, Watanabe M.
  • Organizer: The 51th annual meeting of the European association for the study of the liver (EASL The International Liver Congress 2016)
  • Place of Presentation: Barcelona, Spain
  • Year and Date: 2016-04-13 – 2016-04-17
  • Invited