Hepatic Differentiation of Embryonic Stem Cells by Murine Fetal Liver Mesenchymal Cells

Part of the Methods in Molecular Biology book series (MIMB, volume 946)


Hepatocytes derived from embryonic stem cells (ESCs) are a potential cell source for regenerative medicine. However, it has been technically difficult to differentiate ESCs into mature hepatocytes because the definitive growth factors and molecular mechanisms governing hepatocyte differentiation have not yet been well defined. The CD45CD49f+/−Thy1+gp38+ mesenchymal cells that reside in murine fetal livers induce hepatic progenitor cells to differentiate into mature hepatocytes by direct cell–cell contact. Utilizing these cells, we employ a two-step procedure for hepatic maturation of ESCs: first, ESCs are differentiated into endodermal cells or hepatic progenitor cells, and second, ESC-derived endodermal cells are matured into functional hepatocytes by coculture with murine fetal liver mesenchymal cells. The ESC-derived hepatocyte-like cells possess hepatic functions, including ammonia removal activity, albumin secretion ability, glycogen synthesis and storage, and cytochrome P450 enzymatic activity.

Key words

Embryonic stem cell Fetal liver Hepatocyte Hepatic progenitor cell Mesenchymal cell Thy1 gp38 


  1. 1.
    Hoppo T, Fujii H, Hirose T, Yasuchika K, Azuma H, Baba S, Naito M, Machimoto T, Ikai I (2004) Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver. Hepatology 39:1362–1370PubMedCrossRefGoogle Scholar
  2. 2.
    Kamo N, Yasuchika K, Fujii H, Hoppo T, Machimoto T, Ishii T, Fujita N, Tsuruo T, Yamashita JK, Kubo H, Ikai I (2007) Two populations of Thy1-positive mesenchymal cells regulate in vitro maturation of hepatic progenitor cells. Am J Physiol Gastrointest Liver Physiol 292:G526–G534PubMedCrossRefGoogle Scholar
  3. 3.
    Ishii T, Yasuchika K, Fujii H, Hoppo T, Baba S, Naito M, Machimoto T, Kamo N, Suemori H, Nakatsuji N, Ikai I (2005) In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes. Exp Cell Res 309:68–77PubMedCrossRefGoogle Scholar
  4. 4.
    Ishii T, Yasuchika K, Fukumitsu K, Kawamoto T, Kawamura-Saitoh M, Amagai Y, Ikai I, Uemoto S, Kawase E, Suemori H, Nakatsuji N (2010) In vitro hepatic maturation of human embryonic stem cells by using a mesenchymal cell line derived from murine fetal livers. Cell Tissue Res 339:505–512PubMedCrossRefGoogle Scholar
  5. 5.
    Fukumitsu K, Ishii T, Yasuchika K, Amagai Y, Kawamura-Saito M, Kawamoto T, Kawase E, Suemori H, Nakatsuji N, Ikai I, Uemoto S (2009) Establishment of a cell line derived from a mouse fetal liver that has the characteristic to promote the hepatic maturation of mouse embryonic stem cells by a coculture method. Tissue Eng Part A 15:3847–3856PubMedCrossRefGoogle Scholar
  6. 6.
    Sumi T, Tsuneyoshi N, Nakatsuji N, Suemori H (2007) Apoptosis and differentiation of human embryonic stem cells induced by sustained activation of c-Myc. Oncogene 26:5564–5576PubMedCrossRefGoogle Scholar
  7. 7.
    Suemori H, Yasuchika K, Hasegawa K, Fujioka T, Tsuneyoshi N, Nakatsuji N (2006) Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage. Biochem Biophys Res Commun 345:926–932PubMedCrossRefGoogle Scholar
  8. 8.
    Ishii T, Fukumitsu K, Yasuchika K, Adachi K, Kawase E, Suemori H, Nakatsuji N, Ikai I, Uemoto S (2008) Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells. Am J Physiol Gastrointest Liver Physiol 295:G313–G321PubMedCrossRefGoogle Scholar
  9. 9.
    Yasuchika K, Hirose T, Fujii H, Oe S, Hasegawa K, Fujikawa T, Azuma H, Yamaoka Y (2002) Establishment of a highly efficient gene transfer system for mouse fetal hepatic progenitor cells. Hepatology 36:1488–1497PubMedGoogle Scholar
  10. 10.
    Ishii T, Yasuchika K, Machimoto T, Kamo N, Komori J, Konishi S, Suemori H, Nakatsuji N, Saito M, Kohno K, Uemoto S, Ikai I (2007) Transplantation of embryonic stem cell-derived endodermal cells into mice with induced lethal liver damage. Stem Cells 25:3252–3260PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Takamichi Ishii
    • 1
  • Kentaro Yasuchika
    • 1
  • Iwao Ikai
    • 1
    • 2
  1. 1.Department of Surgery, Graduate School of MedicineKyoto UniversityKyotoJapan
  2. 2.Department of Surgery, Kyoto Medical CenterNational Hospital OrganizationKyotoJapan

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