肝幹細胞分化を基軸としたＨＢＶ ｃｃｃＤＮＡの完全排除を目指す新規治療の創成

2014 Fiscal Year Annual Research Report

• Project/Area Number: 25670366
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: 朝比奈 靖浩 東京医科歯科大学, 医歯(薬)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, 医歯(薬)学総合研究科, 寄附講座講師 (30372444) ; 中川 美奈 東京医科歯科大学, 医歯学融合教育支援センター, 准教授 (30401342)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: HBV / cccDNA / 幹細胞分化 / iPS細胞

Outline of Annual Research Achievements

1．ヒトiPS細胞由来肝幹・前駆細胞および成熟肝細胞の誘導・培養系の確立：HBVは成熟肝細胞でのみで複製するとされるが、定常状態の肝細胞が一定の速度でturn overしているにも関わらず、複製中間体のcccDNAが核内に残存し続ける原因は不明であるため、本研究では、より未分化な幹細胞類似形質を持つ細胞においてcccDNAが保存されることが一因であるかを解明するために、細胞分化度を調節し得る培養系を確立した。即ち、肝細胞分化度の変化がHBV感染サイクルに与える影響を解析するために、ヒトiPS細胞株にactivin A、FGF、BMP4、HGF を段階的に添加することにより肝細胞系譜へと分化誘導した後、これらのうち肝幹細胞マーカーCD13+CD133+陽性細胞を繰り返しFACSにより分離することで、AFP、HNF4aを高度に発現するより純化されたiPS由来肝幹・前駆細胞 (iPS-Hep細胞) の培養法を確立し、一定期間の継代培養を可能とした。またOSM・細胞外マトリクス等の刺激により成熟肝細胞への分化が誘導された細胞の培養法を構築した。

2．iPS-HepにおけるHBVの感染性の検討：純化したiPS-Hep細胞ではNTCPのmRNA発現が肝癌細胞株と比較して100倍以上の高発現していた。本iPS-Hep細胞を用いて種々の条件下でHepG2.2.15細胞由来の培養上清を用いてHBV感染実験を行い、とくに感染効率向上のため dimethyl sulfoxide処理およびpolyethylene glycol添加を行った。HepG2.2.15細胞由来HBVの感染12日後に細胞内に0.3copies/cellのcccDNAと2×108copies/mlのHBVDNAおよぎ上清中に約２ｘ10４copies/ce//のHBVが確認され、成熟分化した肝細胞より分化度の低いiPS-Hep細胞におけるHBV感染が示唆された。

Research Products

• [Journal Article] Impaired induction of IL28B and expression of IFNλ4 associated with non-response to interferon-based therapy in chronic hepatitis C. (2015)
  • Author(s): Murakawa M*, Asahina Y*, Nakagawa M, Sakamoto N, Nitta S, Kusano-Kitazume A, Watanabe T, Kawai-Kitahata F, Otani S, Taniguchi M, Goto F, Nishimura-Sakurai Y, Itsui Y, Azuma S, Kakinuma S, Watanabe M.
  • Journal Title: J Gastroenterol Hepatol Volume: - Pages: -
  • DOI: 10.1111/jgh.12902
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Serum granulysin levels as a predictor of serious telaprevir-induced dermatological reactions. (2015)
  • Author(s): Suda G, Yamamoto Y, Nagasaka A, Furuya K, Kudo M, Yoshimichi C, Tsukuda Y, Tsunematsu S, Sato F, Terasita K, Nakai M, Horimoto H, Sho T, Natsuizka M, Ogawa K, Ohnishi S, Chuma M, Fujita Y, Abe R, Taniguchi M, Nakagawa M, Asahina Y, Sakamoto N.
  • Journal Title: Hepatol Res Volume: - Pages: -
  • DOI: 10.1111/hepr.12421
  • Peer Reviewed
• [Journal Article] Hepatocellular carcinoma risk assessment using gadoxetic acid-enhanced hepatocyte phase magnetic resonance imaging. (2015)
  • Author(s): Komatsu N, Motosugi U, Maekawa S, Shindo K, Sakamoto M, Sato M, Tatsumi A, Miura M, Amemiya F, Nakayama Y, Inoue T, Fukasawa M, Uetake T, Ohtaka M, Sato T, Asahina Y, Kurosaki M, Izumi N, Ichikawa T, Araki T, Enomoto N.
  • Journal Title: Hepatol Res Volume: - Pages: -
  • DOI: 10.1111/hepr.12309
  • Peer Reviewed
• [Journal Article] Changes in plasma vascular endothelial growth factor at 8 weeks after sorafenib administration as predictors of survival for advanced hepatocellular carcinoma. (2014)
  • Author(s): Tsuchiya K*, Asahina Y*, Matsuda S, Muraoak M, Nakata T, Suzuki Y, Tamaki N, Yasui Y, Suzuki S, Hosokawa T, Nishimura T, Ueda K, Kuzuya T, Nakanishi H, Itakura J, Takahashi Y, Kurosaki M, Enomoto N, Izumi N.
  • Journal Title: Cancer Volume: 120 Pages: 229-273
  • DOI: 10.1002/cncr.28384
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Risk factors for exceeding the Milan criteria after successful radiofrequency ablation in patients with early stage hepatocellular carcinoma. (2014)
  • Author(s): Tsuchiya K, Asahina Y, Tamaki N, Yasui Y, Hosokawa T, Ueda K, Nakanishi H, Itakura J, Kurosaki M, Enomoto N, Izumi N.
  • Journal Title: Liver Transpl Volume: 20 Pages: 291-297
  • Peer Reviewed
• [Journal Article] Reduced organic anion transporter expression is a risk factor for hepatocellular carcinoma in chronic hepatitis C patients: A propensity score matching study. (2014)
  • Author(s): Yasui Y, Kudo A, Kurosaki M, Matsuda S, Muraoka M, Tamak Ni, Suzuki S, Hosokawa T, Ueda K, Matsunaga K, Nakanishi H, Tsuchiya K, Itakura J, Takahashi Y, Tanaka S, Asahina Y, Enomoto N, Arii S, Izumi N.
  • Journal Title: Oncology Volume: 86 Pages: 53-62
  • DOI: 10.1159/000356643
  • Peer Reviewed
• [Journal Article] Susceptibility and Resistance HLA-DP Alleles to HBV-Related Diseases Identified by a Trans-Ethnic Association Study in Asia. (2014)
  • Author(s): Nishida N, Sawai H, Asahina Y, Mizokami M, et al.
  • Journal Title: PLoS One Volume: - Pages: -
  • DOI: 10.1371/journal.pone.0086449
  • Peer Reviewed
• [Journal Article] Impaired brain activity in cirrhotic patients with minimal hepatic encephalopathy: evaluation by near infrared spectroscopy. (2014)
  • Author(s): Nakanishi H, Kurosaki M, Nakanishi K, Tsuchiya K, Noda T, Tamaki N, Yasui Y, Hosokawa T, Ueda K, Itakura J, Anami K, Asahina Y, Enomoto N, Higuchi T, Izumi N.
  • Journal Title: Hepatol Res Volume: 44 Pages: 319-326
  • DOI: 10.1111/hepr.12127
  • Peer Reviewed
• [Journal Article] Prospective comparison of real-time tissue elastography and serum fibrosis markers for the estimation of liver fibrosis in chronic hepatitis C patients. (2014)
  • Author(s): Tamaki N, Kurosaki M, Matsuda S, Nakata T, Muraoka M, Suzuki Y, Yasui Y, Suzuki S, Hosokawa T, Nishimura T, Ueda K, Tsuchiya K, Nakanishi H, Itakura J, Takahashi Y, Matsunaga K, Taki K, Asahina Y, Izumi N.
  • Journal Title: Hepatol Res Volume: 44 Pages: 720-727
  • DOI: 10.1111/hepr.12179
  • Peer Reviewed
• [Journal Article] JSH guidelines for the management of hepatitis B virus infection. (2014)
  • Author(s): Asahina Y, HayashinN, Hiramatsu N, Izumi N, Koike K, Kumada H, Kurosaki M, Oketani M, Suzuki F, Takikawa H, Tanaka A, Tanaka E, Tanaka Y, Tsubouchi H, Yotsuyanagi H.
  • Journal Title: Hepatol Res Volume: 44 Pages: 1-58
  • DOI: 10.1111/hepr.12269
  • Peer Reviewed
• [Journal Article] JSH guidelines for the management of hepatitis C virus infection: a 2014 update for genotype 1. (2014)
  • Author(s): Asahina Y, HayashinN, Hiramatsu N, Izumi N, Koike K, Kumada H, Kurosaki M, Oketani M, Suzuki F, Takikawa H, Tanaka A, Tanaka E, Tanaka Y, Tsubouchi H, Yotsuyanagi H.
  • Journal Title: Hepatol Res Volume: 44 Pages: 59-70
  • DOI: 10.1111/hepr.12272
  • Peer Reviewed
• [Presentation] Gene Alterations in TERT promoter, CTNNB1, and TP53 are closely associated with development and prognosis of hepatocellular carcinoma: comprehensive analyses by next generation sequencing technology. (2015)
  • Author(s): Asahina Y, Kawai-Kitahata F, Kaneko S, Nagata H, Goto F, Otani S, Taniguchi M, Murakawa M, Nitta S, Watanabe T, Tasaka-Fujita M, Itsui Y, Nakagawa M, Kakinuma S, Tanaka S, Tanabe M, Enomoto N, Watanabe M.
  • Organizer: The 50th annual meeting of the European association for the study of the liver (EASL The International Liver Congress 2015)
  • Place of Presentation: Vienna, Austria
  • Year and Date: 2015-04-23
• [Presentation] Gene alterations in β-catenin and p53/ cell cycle control pathway are closely associated with development and prognosis of hepatocellular carcinoma: Comprehensive analyses by next generation sequencing technology. (2014)
  • Author(s): Kawai-Kitahata F, Asahina Y, Kaneko S, Nagata H, Goto F, Otani S, Taniguchi M, Murakawa M, Nitta S, Watanabe T, Tasaka-Fujita M, Itsui Y, Nakagawa M, Kakinuma S, Enomoto N, Watanabe M.
  • Organizer: The 65th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2014)
  • Place of Presentation: Boston, USA
  • Year and Date: 2014-11-07 – 2014-11-11
• [Presentation] Matrix Metalloproteinase-14 regulates the maturation of fetal hepatic stem/progenitor cells in mice. (2014)
  • Author(s): Otani S, Kakinuma S, Kamiya A, Goto F, Kaneko S, Azuma S, Asahina Y, Watanabe M.
  • Organizer: The 65th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2014)
  • Place of Presentation: Boston, USA
  • Year and Date: 2014-11-07 – 2014-11-11
• [Presentation] Impact of host and therapeutic factors and resistant associated variants on response to interferon based- direct acting antiviral treatment in difficult-to-treat chronic hepatitis C patients. (2014)
  • Author(s): Nakagawa M, Asahina Y, Taniguchi M, Watanabe T, Nishimura-Sakurai Y, Itsui Y, Azuma S, Kakinuma S, Tanaka Y, Watanabe M.
  • Organizer: The 65th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2014)
  • Place of Presentation: Boston, USA
  • Year and Date: 2014-11-07 – 2014-11-11
• [Presentation] Expression of IFN/.4 in liver and PBMC is closely associated with higher basal expression of ISGs and impaired induction of IL28B by interferon treatment in chronic hepatitis C non-responder patients. (2014)
  • Author(s): Murakawa M, Asahina Y, Nakagawa M, Sakamoto N, Nitta S, Kawai-Kitahata F, Taniguchi M, Watanabe T, Itsui Y, Kakinuma S, Watanabe M.
  • Organizer: The 65th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2014)
  • Place of Presentation: Boston, USA
  • Year and Date: 2014-11-07 – 2014-11-11
• [Presentation] Emergence or selection of resistant associated variant immediately after initiation of the therapy is predictive for failure of direct acting antiviral therapy: ultra-deep sequencing analyses for serial time points. (2014)
  • Author(s): Watanabe T, Asahina Y, Nakagawa M, Kakinuma S, YItsui Y, Taniguchi T, Murakawa M, Nagata H, Miura M, Maekawa S, Enomoto E, Watanabe M.
  • Organizer: The 65th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2014)
  • Place of Presentation: Boston, USA
  • Year and Date: 2014-11-07 – 2014-11-11
• [Presentation] Clinical outcomes in patients who develop hepatocellular carcinoma after hepatitis C viral eradication by antiviral therapy. (2014)
  • Author(s): Tsuchiya K, Yasui Y, Tamaki N, Suzuki S, Hosokawa T, Nakanishi H, Itakura J, Takahashi Y, Kurosaki M, Asahina Y, Izumi N.
  • Organizer: The 65th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2014)
  • Place of Presentation: Boston, USA
  • Year and Date: 2014-11-07 – 2014-11-11
• [Presentation] Associations of HLA-DPB1 with CHB infection and HBV related HCC in Asia. (2014)
  • Author(s): Nishida N, Sawai H, Asahina Y, Mizokami M, et al.
  • Organizer: The 65th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2014)
  • Place of Presentation: Boston, USA
  • Year and Date: 2014-11-07 – 2014-11-11
• [Presentation] Impaired IL28B gene induction and expression of IFNλ4 influenced by the polymorphisms near IL28B gene are closely associated with a non-response to interferon in chronic hepatitis C patients. (2014)
  • Author(s): Asahina Y, Murakawa M, NittaS, Itsui Y, Nakagawa M, Azuma S, Kakinuma S, Watanabe M.
  • Organizer: The 49th annual meeting of the European association for the study of the liver (EASL The International Liver Congress 2014)
  • Place of Presentation: London, UK
  • Year and Date: 2014-04-08 – 2014-04-14
• [Presentation] Gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid magnetic resonance imaging and contrast enhanced ultrasonography with sonazoid as part of therapeutic strategies for small nonhypervascular hepatic nodular lesions. (2014)
  • Author(s): Tsuchiya K, Yasui Y, Takada N, Nakakuki S, Matsuda S, Kaneko S, Muraoka M, Yamashita N, Hattori N, Tamaki N, Osaki S, Suzuki T, Hosokawa K, Ueda K, Nakanishi H, Itakura J, Takahashi Y, Kurosaki M, Asahina Y, Izumi N.
  • Organizer: The 49th annual meeting of the European association for the study of the liver (EASL The International Liver Congress 2014)
  • Place of Presentation: London, UK
  • Year and Date: 2014-04-08 – 2014-04-14