Advertisement

Direct and indirect coculture of mouse hepatic progenitor cells with mouse embryonic fibroblasts for the generation of hepatocytes and cholangiocytes

  • Tao Zhou
  • Wei Wang
  • Yasen Aimaiti
  • Xin Jin
  • Zhixin Chen
  • Liang Chen
  • Dewei Li
Original Article
  • 25 Downloads

Abstract

The widespread use of hepatocytes and cholangiocytes for regenerative medicine and tissue engineering is restricted by the limited number of hepatocytes and cholangiocytes; a simple and effective method for the expansion and differentiation of the hepatic progenitor cells (HPCs) is required. Recent studies demonstrated that mouse embryonic fibroblasts (MEFs) play an important role in supporting the proliferation of the mouse hepatic progenitor cells (mHPCs). However, the effect of direct and indirect coculture of MEFs with mHPCs on the differentiation of mHPCs is poorly studied. Herein, we show that mHPCs rapidly proliferate and form colonies in direct or indirect contact coculture with MEFs in the serum-free medium. Importantly, after direct contact coculture of the mHPCs with MEFs for 6 days, mHPCs expressed the hepatic marker albumin (ALB) and did not express the cholangiocyte marker CK19, indicating their differentiation into hepatocytes. In contrast, after indirect contact coculture of the mHPCs with MEFs for 6 days, mHPCs expressed the cholangiocyte marker CK19 and did not express the hepatic marker ALB, indicating their differentiation into cholangiocytes. These results indicate that direct and indirect contact cocultures of the mHPCs with MEFs are useful for rapidly producing hepatocytes and cholangiocytes.

Keywords

Hepatic progenitor cells Mouse embryonic fibroblasts Coculture Differentiation 

Abbreviations

MEFs

Mouse embryonic fibroblasts

mHPCs

Mouse hepatic progenitor cells

HPCs

Hepatic progenitor cells

FACS

Fluorescence-activated cell sorting

DPBS

Dubecco’s phosphate buffered saline

Dlk

Delta-like 1 homologue

ALB

Albumin

Notes

Funding

This work was supported by the General Program of National Natural Science Foundation of China (Grant No. 81470898).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interests.

References

  1. Alwahsh SM, Rashidi H, Hay DC (2018) Liver cell therapy: is this the end of the beginning? Cell Mol Life Sci 75:1307–1324CrossRefGoogle Scholar
  2. Anzai K, Chikada H, Tsuruya K, Ida K, Kagawa T et al (2016) Foetal hepatic progenitor cells assume a cholangiocytic cell phenotype during two-dimensional pre-culture. Sci Rep 6:28283CrossRefGoogle Scholar
  3. Asumda FZ, Hatzistergos KE, Dykxhoorn DM, Jakubski S, Edwards J et al (2018) Differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules. Differentiation 101:16–24CrossRefGoogle Scholar
  4. Bhandari RN, Riccalton LA, Lewis AL, Fry JR, Hammond AH, Tendler SJB et al (2001) Liver tissue engineering: a role for co-culture systems in modifying hepatocyte function and viability. Tissue Eng 7:345–357CrossRefGoogle Scholar
  5. Boulter L, Lu WY, Forbes SJ (2013) Differentiation of progenitors in the liver: a matter of local choice. J Clin Investig 123:1867–1873CrossRefGoogle Scholar
  6. Dianat N, Dubois-Pot-Schneider H, Steichen C, Desterke C, Leclerc P et al (2014) Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells. Hepatology 60:700–714CrossRefGoogle Scholar
  7. Gordillo M, Evans T, Gouon-Evans V (2015) Orchestrating liver development. Development 142:2094–2108CrossRefGoogle Scholar
  8. Hoppo T, Fujii H, Hirose T, Yasuchika K, Azuma H et al (2004) Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver. Hepatology 39:1362–1370CrossRefGoogle Scholar
  9. Hu C, Li L (2015) In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration. Protein Cell 6:562–574CrossRefGoogle Scholar
  10. Ito H, Kamiya A, Ito K, Yanagida A, Okada K et al (2012) In vitro expansion and functional recovery of mature hepatocytes from mouse adult liver. Liver Int 32:592–601CrossRefGoogle Scholar
  11. Ito K, Yanagida A, Okada K, Yamazaki Y, Nakauchi H et al (2014) Mesenchymal progenitor cells in mouse foetal liver regulate differentiation and proliferation of hepatoblasts. Liver Int 34:1378–1390CrossRefGoogle Scholar
  12. Jiang G, Wan X, Wang M, Zhou J, Pan J et al (2016) A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers. Cytotechnology 68:1603–1614CrossRefGoogle Scholar
  13. Kamiya A, Inagaki Y (2015) Stem and progenitor cell systems in liver development and regeneration. Hepatol Res 45:29–37CrossRefGoogle Scholar
  14. Kamiya A, Ito K, Yanagida A, Chikada H, Iwama A et al (2015) MEK-erk activity regulates the proliferative activity of fetal hepatoblasts through accumulation of p16/19(cdkn2a). Stem Cells Dev 24:2525–2535CrossRefGoogle Scholar
  15. Liu Y, Li H, Yan S, Wei J, Li X (2014) Hepatocyte cocultures with endothelial cells and fibroblasts on micropatterned fibrous mats to promote liver-specific functions and capillary formation capabilities. Biomacromolecules 15:1044–1054CrossRefGoogle Scholar
  16. Nagai H, Terada K, Watanabe G, Ueno Y, Aiba N et al (2002) Differentiation of liver epithelial (stem-like) cells into hepatocytes induced by coculture with hepatic stellate cells. Biochem Biophys Res Commun 293:1420–1425CrossRefGoogle Scholar
  17. Naoki Tanimizu MN, Saito Hiroki, Tsujimura Tohru, Miyajima Atsushi (2003) Isolation of hepatoblasts based on the expression of Dlk/Pref-1. J Cell Sci 116:1775–1786CrossRefGoogle Scholar
  18. Nicolas CT, Hickey RD, Chen HS, Mao SA, Lopera Higuita M et al (2017) Concise review: liver regenerative medicine—from hepatocyte transplantation to bioartificial livers and bioengineered grafts. Stem Cells 35:42–50CrossRefGoogle Scholar
  19. Oertel M (2011) Fetal liver cell transplantation as a potential alternative to whole liver transplantation? J Gastroenterol 46:953–965CrossRefGoogle Scholar
  20. Ogawa M, Ogawa S, Bear CE, Ahmadi S, Chin S et al (2015) Directed differentiation of cholangiocytes from human pluripotent stem cells. Nat Biotechnol 33:853–861CrossRefGoogle Scholar
  21. Okada K, Kamiya A, Ito K, Yanagida A, Ito H et al (2012) Prospective isolation and characterization of bipotent progenitor cells in early mouse liver development. Stem Cells Dev 21:1124–1133CrossRefGoogle Scholar
  22. Onitsuka I, Tanaka M, Miyajima A (2010) Characterization and functional analyses of hepatic mesothelial cells in mouse liver development. Gastroenterology 138:1525–1535CrossRefGoogle Scholar
  23. Paschos PK, Brown WE, Eswaramoorthy R, Hu JC, Athanasiou KA (2015) Advances in tissue engineering through stem cell-based co-culture. J Tissue Eng Regen Med 9:488–503CrossRefGoogle Scholar
  24. Si-Tayeb K, Lemaigre FP, Duncan SA (2010) Organogenesis and development of the liver. Dev Cell 18:175–189CrossRefGoogle Scholar
  25. Suzuki K, Tanaka M, Watanabe N, Saito S, Nonaka H et al (2008) p75 neurotrophin receptor is a marker for precursors of stellate cells and portal fibroblasts in mouse fetal liver. Gastroenterology 135:e273Google Scholar
  26. Takayama KAN, Mimura N, Akahira R, Taniguchi Y, Ikeda M, Sakurai F, Ohara O, Morio T, Sekiguchi K, Mizuguchi H (2017) Generation of safe and therapeutically effective human induced pluripotent stem cell-derived hepatocyte-like cells for regenerative medicine. Hepatol Commun 1:1058–1069CrossRefGoogle Scholar
  27. Tsuruya K, Chikada H, Ida K, Anzai K, Kagawa T et al (2015) A paracrine mechanism accelerating expansion of human induced pluripotent stem cell-derived hepatic progenitor-like cells. Stem Cells Dev 24:1691–1702CrossRefGoogle Scholar
  28. Wang W, Lü F, Jin X, Dewei LI (2017) Isolation and amplification of hepatic progenitor cells from fetal mouse in vitro. Basic Clin Med 37:648–652CrossRefGoogle Scholar
  29. Wang W, Feng Y, Aimaiti Y, Jin X, Mao X et al (2018) TGFβ signaling controls intrahepatic bile duct development may through regulating the Jagged1-Notch-Sox9 signaling axis. J Cell Physiol 233:5780–5791CrossRefGoogle Scholar
  30. Weissman ILAD, Gage F (2001) Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations. Annu Rev Cell Dev Biol 17:387–403CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Hepatobiliary SurgeryThe First Affiliated Hospital of Chongqing Medical UniversityChongqingPeople’s Republic of China

Personalised recommendations