iPS細胞由来肝細胞系譜におけるHBV感染系の可視化とHBVを根絶する治療の創成

2015 Fiscal Year Research-status 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, 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)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Keywords: HBV / iPS細胞 / 肝幹・前駆細胞 / 幹細胞分化

Outline of Annual Research Achievements

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、核酸アナログによるHBV複製抑制下において複製中間体のcccDNAが核内に残存し続ける原因は不明である。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とともに、従来の薬剤開発とは視点を異にする細胞分化機構に着目した新規治療の創出に新たな知的・技術的基盤を確立することを目的として、これまでに分化誘導・純化法を確立した継代可能なヒトiPS細胞由来肝幹･前駆細胞(iPS-HPC)及び成熟化肝細胞株(iPS-Hep)において研究を進めた。iPS由来細胞におけるHBV感染については、より高感度かつ簡便に検出するために、新たに開発された蛍光色素組み換えHBVを用いて検討を行った。より未分化な形質を持つiPS-HPCはNTCPを発現しており、HBV添加12日後に100倍以上のluciferase活性を示し、細胞内cccDNAおよび上清中の抗原量の増加が認められた。一方、より成熟した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。本培養系におけるIFNα添加時のIFN誘導遺伝子（MxA, PKR, ISG15)の発現量誘導能は、肝癌由来細胞株であるHepG2－NTCPに比し有意に高く(p<0.001)、 IFNαによる抗原産生の有意な低下とPreS1蛋白による阻害効果が認められた。以上より、より低分化度のiPS-HPCにおいてHBV感染とcccDNA産生が確認されたとともに、iPS-HPC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。

Current Status of Research Progress

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

Reason

蛍光組み換えHBVをiPS細胞由来肝細胞系譜の感染実験に導入し、効率的にHBV感染を定量可能とし、細胞分化とHBV感染の関連を明らかとした。更に、本系を用いることでより生理的状態に近い細胞で薬剤の感受性が検討可能なことを示した。

Strategy for Future Research Activity

細胞分化の違いによるHBV感染・増殖の差に、如何なる因子が関与するかを解明し、その分子機構が新たな治療標的となり得るかを検討する。また、継代可能なiPS-HPCにおいて感染効率の高いHBV培養増殖系を開発するために、iPS細胞における遺伝子導入技術を駆使し、本系を改良を試みる。

Causes of Carryover

試薬等が計画当初より廉価で購入可能であったため。

Expenditure Plan for Carryover Budget

検討する数・種類を拡大して解析を行うため、試薬を増量して購入する予定である。

Research Products

• [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] 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
• [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: doi: 10.1016/j.bbrc.2015.12.105
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Genetic polymorphisms of IL28B and PNPLA3 are predictive for HCV related rapid fibrosis progression and identify patients who require urgent antiviral treatment with new regimens. (2015)
  • Author(s): Tamaki N, Kurosaki M, Higuchi M, Takada T, Nakakuki N, Yasui Y, Suzuki S, Tsuchiya K, Nakanishi H, Itakura J, Takahashi Y, Ogawa S, Tanaka Y, Asahina Y, Izumi N
  • Journal Title: PLoS One Volume: 10 Pages: e0137351
  • DOI: doi: 10.1371/journal.pone.0137351
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Amino acid 1 polymorphisms in hepatitis C virus core affect infectious virus production and major histocompatibility complex class I molecule expression. (2015)
  • Author(s): Tasaka-Fujita M, Sugiyama N, Kang W, Masaski T, Murayama A, Yamada N, Sugiyama R, Tsukuda S, Watashi K, Asahina Y, Sakamoto N, Wakita T, Shin E-C, Kato T
  • Journal Title: Sci Rep Volume: 5 Pages: 13994
  • DOI: doi: 10.1038/srep13994
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] Hepatocarcinogenesis after hepatitis C viral eradication. (2016)
  • Author(s): Asahina Y
  • Organizer: The 10th Asian Pacific Association for the Study of the Liver (APASL) Single Topic Conference
  • Place of Presentation: Kaoshiung（Taiwan）
  • Year and Date: 2016-06-08 – 2016-06-10
  • Int'l Joint Research / Invited
• [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 S, 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 – 2016-04-18
  • Int'l Joint Research
• [Presentation] Comprehensive analyses of mutations and hepatitis B virus integration in hepatocellular carcinoma. (2016)
  • Author(s): Kawai-Kitahata F, Asahina Y, Tanaka S, Kakinuma S, Murakawa M, Nitta S, Otani S, Goto F, Nagata H, Kaneko S, Asano Y, Miyoshi M, Tsunoda T, Azuma S, Itsui Y, Nakagawa M, Tanabe M, Maekawa S, Enomoto N, Watanabe M
  • Organizer: 25th conference of the Asian Pacific Association for the Study of the Liver (APASL 2016)
  • Place of Presentation: グランドプリンスホテル新高輪（東京都港区）
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [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 S, Watanabe M.
  • Organizer: 25th conference of the Asian Pacific Association for the Study of the Liver (APASL 2016)
  • Place of Presentation: グランドプリンスホテル新高輪（東京都港区）
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [Presentation] Serial changes in M2BPGi levels as predictors of fibrosis and HCC in chronic hepatitis C. (2016)
  • Author(s): Nagata H, Sakurai Y, Nakagawa M, Kakinuma S, Azuma S, Murakawa M, Nitta S, Itsui Y, Asahina Y, Watanabe M
  • Organizer: 25th conference of the Asian Pacific Association for the Study of the Liver (APASL 2016)
  • Place of Presentation: グランドプリンスホテル新高輪（東京都港区）
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [Presentation] In vitro characterization and drug sensitivity analysis for HCV NS5A resistance-associated variants. (2016)
  • Author(s): Nitta S, Asahina Y, Wakita T, Kato T
  • Organizer: 25th conference of the Asian Pacific Association for the Study of the Liver (APASL 2016)
  • Place of Presentation: グランドプリンスホテル新高輪（東京都港区）
  • Year and Date: 2016-02-20 – 2016-02-24
  • Int'l Joint Research
• [Presentation] Human induce pluripotent stem cell-dereived hepatic progenitor-like cells and hepatocyte-like cells as a model for interaction between hepatitis B virus and host cells. (2015)
  • Author(s): Kaneko S, Kakinuma S, Asahina Y, Kamiya A, Nitta S, Tsunoda T, Miyosi M, Nagata H, Goto F, Otani S, Murakawa M, Kawai-Kitahata F, Itsui Y, Nakagawa M, Azuma S, Watanabe M
  • Organizer: The 66th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2015)
  • Place of Presentation: San Francisco（USA）
  • Year and Date: 2015-11-13 – 2015-11-17
  • Int'l Joint Research
• [Presentation] Comprehensive analyses of mutations and hepatitis B virus integration in hepatocellular carcinoma with clinicopathological features. (2015)
  • Author(s): Kawai-Kitahata F, Asahina Y, Tanaka S, Kakinuma S, Murakawa M, Nitta S, Watanabe T, Otani S, Goto F, Nagata H, Kaneko S, Azuma S, Itsui Y, Nakagawa M, Tanabe M, Maekawa S, Enomoto N, Watanabe M
  • Organizer: The 66th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2015)
  • Place of Presentation: San Francisco（USA）
  • Year and Date: 2015-11-13 – 2015-11-17
  • Int'l Joint Research
• [Presentation] Effects of resistance mutations of NS5A inhibitor on viral production and susceptibility to anti-HCV reagents in recombinant hepatitis C viruses with NS5A of genotype 1b. (2015)
  • Author(s): Nitta S, Asahina Y, Wakita T, Kato T
  • Organizer: The 66th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2015)
  • Place of Presentation: San Francisco（USA）
  • Year and Date: 2015-11-13 – 2015-11-17
  • Int'l Joint Research
• [Presentation] Expression of IFN lambda 4 in liver is closely associated with non-response to antiviral therapy through the regulation of basal expression of ISGs in chronic hepatitis C patients but not in hepatitis B. (2015)
  • Author(s): Murakawa M, Asahina Y, Kawai-Kitahata F, Nagata H, Kaneko S, Nitta S, Watanabe T, Itsui Y, Nakagawa M, Kakinuma S, Iijima S, Tanaka Y, Watanabe M, Tanaka Y
  • Organizer: The 66th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2015)
  • Place of Presentation: San Francisco（USA）
  • Year and Date: 2015-11-13 – 2015-11-17
  • Int'l Joint Research
• [Presentation] Serial change of resistant associated variants during early phase of NS3/4A triple therapy and the final virological outcome: analyses by ultra-deep sequenceing technology. (2015)
  • Author(s): Watanabe T, Asahina Y, Nakagawa M, Kakinuma S, Itsui Y, Nagata H, Murakawa M, Kawai-Kitahata F, Miura M, Maekawa S, Enomoto N, Watanabe M
  • Organizer: The 66th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2015)
  • Place of Presentation: San Francisco（USA）
  • Year and Date: 2015-11-13 – 2015-11-17
  • Int'l Joint Research
• [Presentation] Variations of the genome and interaction of hepatitis B viral X protein associated with hepatocarcinogenesis. (2015)
  • Author(s): Nagata H, Itsui Y, Kawai-Kitahata F, Kaneko S, Murakawa M, Nitta S, Nakagawa M, Azuma S, Kakinuma S, Asahina Y
  • Organizer: The 66th Annual Meeting of the American Association for the Study of Liver Disease (AASLD The Liver Meeting 2015)
  • Place of Presentation: San Francisco（USA）
  • Year and Date: 2015-11-13 – 2015-11-17
  • Int'l Joint Research