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Research for the in vitro reconstruction of hepatic tissues : an application for an artificial liver.

Research Project

Project/Area Number 08670260
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Experimental pathology
Research InstitutionSapporo Medical University School of Medicine

Principal Investigator

MITAKA Toshihiro  Department of Pathology, Cancer Research Institute, Sapporo Medical University Assistant Professor, がん研究所, 講師 (50231618)

Co-Investigator(Kenkyū-buntansha) MOCHIZUKI Yohichi  Department of Pathology, Cancer Research Institute, Sapporo Medical University P, がん研究所, 教授 (40045381)
Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
Keywordssmall hepatocytes / three-dimensional structure / stem cells / proliferation / differentiation / hepatic nonparenchymal cells / extracellular matrix / 細胞外期質 / 肝細胞 / ラット / ヒト / 転写因子
Research Abstract

A small hepatocyte can clonally proliferate for more than 3 months when the cells are cultured in DMEM supplemented with 10% FBS,10mM nicotinamide, 1mM ascorbic acid 2-phosphate, 10ng/mI EGF and 1% DMSO.The cell divides to form a colony and the number of the cells reaches to more than 100 cells within 20 days. With time in culture, cells with a large cytoplasm appear within a colony. They have many mitochondria and large peroxisomes with a crystaline nucleoid, which are typical mature heatocytes. Immunoreactivity to Cx32 and well-developed bile canaliculus structures are often observed between cells. Thus, we suggest that small hepatocytes may be considered as "committed progenitor cells" that can further differentiate into mature hepatocytes.
We established a culture system in which, by adding 2% DMSO to the culture medium after the hepatocytes proliferate, the cells are able to recover differentiated functions such as albumin and transferrin secretion. Although expressions of connexin 32, Cx 26, and tryptophan 2,3-dioxygenase, thought to a highly differentiated function of mature hepatocytes, have never been maintained or re-induced in primary hepatocytes cultured for long period of time by using the conventional culture methods, those mPNA expressions were gradually restored with time after the addition of 2% DMSO and maintained for a month in this culture system.
We showed the role of the liver-enriched transcription factors in the transition during which proliferating hepatocytes become quiescent. We found that hepatic differentiation requires not only inhibition of DNA synthesis but also induction of appropriate transcription facors. Thus, expression of HNF3gamma, C/EBPalpha, and C/EBPbeta may be necessary for hepatocytes to acquire highly differentiated functions in addition to coexpression of certain amounts of transcripts of HNF1alpha, HNF1beta, HNF3alpha, HNF3beta, and HNF4 as well as suppression of C/EBPdelta.

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • Research Products

    (30 results)

All Other

All Publications (30 results)

  • [Publications] Toshihiro Mitaka, et al.: "Growth and Maturation of Small Hepatocytes" Journal of Gastroenterology and Hepatology. (in press). (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 三高 俊広: "肝の再生:初代培養肝細胞研究から肝再生現象を考える" BIO Clinica. 13(印刷中). (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Toru Mizuguchi, et al.: "Alteration of expression of liver-enriched transcription factors in the transition between growth and differentiation" Journal of Cellular Physiology. 174. 273-284 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Takashi Kojima, et al.: "Different changes in expression and junction of connexin26 and connexin 32 during DNA synthesis and redifferentiation" Hepatology. 26. 585-597 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Takashi Kojima, et al.: "Effect of melatonin on proliferation,oxidative stress and Cx32 gap junction protein expression in primary cultures" Cell Structure and Function. 22. 347-356 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 三高 俊広: "肝臓の多能性幹細胞" Molecular Medicine. 34. 554-565 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Takashi Kojima, et al.: "Formation of actin filament networks in cultured rat hepatocytes treated with DMSO and glucagon" Cell Structure and Function. 22. 269-278 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Toru Mizuguchi, et al.: "Recovery of mRNA expression of tryptophan 2,3-oxygenase and serine dehydratase in the long-term cultures of primary rat" Journal of Biochemistry(Tokyo). 120. 511-517 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Takashi Kojima, et al.: "Changes in cellular distribution of connexins 32 and 26 during formation of gap junctions in primary cultures of rat" Experimental Cell Research. 223. 314-326 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Takashi Kojima, et al.: "Effects of oxygen radical scavengers on connexin 26 and 32 expression in primary cultures of adult rat hepatocytes" Carcinogenesis. 17. 537-544 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Toshihiro Mitaka, etal.: "Subculture of proliferating primary adult rat hepatocytes in medium supplemented with nicotinamide and EGF" In Vitro Cellular and Developmental Biology. 32. 469-477 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Mitaka T,et al.: "Growth and maturation of small hepatocytes (review)" Journal of Gastroenterology and Hepatology. in press. (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Mizuguchic T,et al.: "Alteration of expression of liver-enriched transcription factors in the transition between growth and differentiation of primary cultured rat hepatocytes." Journal of Cellular Physiology. 174. 273-284 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kojima T,et al.: "Different changes in expression and junction of connexin 26 and connexin 32 during DNA synthesis and redifferentiation in primary rat hepatocytes using a DMSO culture system." Hepatology. 26. 585-597 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kojima T,et al.: "Effects of melatonin on proliferation, oxidative stress and Cx32 gap junction protein expression in primary cultures of adult rat hepatocytes." Cell Structure and Function. 22. 347-356 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kojima T,et al.: "Formation of actin filament networks in cultured rat hepatocytes treated with DMSO and glucagon." Cell Structure and Function. 22. 269-278 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Mizuguchic T,et al.: "Recovery of mRNA expression of tryptophan 2,3-oxygenase and serine dehydratase in the long-term cultures of primary rat hepatocytes." Journal of Biochemistry (Tokyo). 120. 511-517 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kojima T,et al.: "Changes in cellular distribution of connexins 32 and 26 during formation of gap junctions in primary cultures of rat hepatocytes." Experimental Cell Research. 222. 314-326 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Kojima T,et al.: "Effects of oxygen radical scavengers on connexin 26 and 32 expression in primary cultures of adult rat hepatocytes." Carcinogenesis. 17. 537-544 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Mitaka T,et al.: "Subculture of proliferating primary adult rat hepatocytes in medium supplemented with nicotinamide and EGF." In Vitro Cellular and Developmental Biology, Animal. 32. 469-477 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Toshihiro Mitaka et al.: "Growth and maturation of small hepatocytes" Journal of Gastroenterology and Hepatology. (in press). (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] 三高 俊広: "肝の再生:初代培養肝細胞研究から肝再生現象を考える" BIO Clinica. (発表予定). (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Toru Mizuguchi et al.: "Alteration of expression of liver-enriched transcription factors in the transition between growth and differentiation" Journal of Cellular Physiology. 174. 273-284 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Manabu Matsuya et al.: "Cell adhesion kinase forms a complex with a new member,Hic-5,of proteins localized at focal adhesions." Journal of Biological Chemistry. 273. 1003-1014 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Takashi Kojima et al.: "Different changes in expression and junction of connexin 26 and connexin 32 during DNA synthesis and redifferentiation" Hepatology. 26. 585-597 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Takashi Kojima et al.: "Formation of actin filament networks in cultured rat hepatocytes treated with DMSO and glucagon" Cell Structure and Function. 22. 269-278 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 三高 俊広: "肝臓の多能性幹細胞" Molecular Medicine. 34(5)(発表予定). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Toshihiro Mitaka: "Restricted Expression of Cell Adhesion Kinase-b in Rat Tissues" American Journal of Pathology. 150(1). 267-281 (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Toshihiro Mitaka: "Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF" In Vitro Cellular and Developmental Biology. 32(8). 469-477 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Toru Mizuguchi: "Recovery of mRNA Expression of Tryptophan 2,3-Dioxygenase and Serine Dehydratase in Long-Term Cultures of Primary Rat Hepatocytes" Journal of Biochemistry (Tokyo). 120(3). 511-517 (1996)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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