Abstract
Hepatocytes derived from human embryonic stem cells (hESCs) are an attractive cell source for regenerative medicine. We previously reported the differentiation of hESCs into alpha-fetoprotein (AFP)-producing endodermal cells by using extracellular matrix and growth factors. We also reported the establishment of the MLSgt20 cell line, which was derived from mesenchymal cells residing in murine fetal livers and accelerated the hepatic maturation of both murine hepatic progenitor cells and murine ESCs. In this study, hESC-derived AFP-producing cells were isolated by using a flow cytometer and co-cultured with MLSgt20 cells. The co-cultured hESC-derived AFP-producing cells had the immunocytological characteristics of hepatocytes, expressed mature hepatocyte markers (as indicated by reverse transcription and the polymerase chain reaction), and displayed higher hepatocyte functions including ammonia removal, cytochrome P450 3A4/7 activity, and the ability to produce and store glycogen. However, the MLSgt20 cells did not directly cause undifferentiated hESCs to mature into hepatocyte-like cells. The co-culture method was thus successfully shown to induce the differentiation of hESC-derived endodermal cells into functional hepatocyte-like cells.
Similar content being viewed by others
References
Cai J, Zhao Y, Liu Y, Ye F, Song Z, Qin H, Meng S, Chen Y, Zhou R, Song X, Guo Y, Ding M, Deng H (2007) Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology 45:1229–1239
Chen SS, Fitzgerald W, Zimmerberg J, Kleinman HK, Margolis L (2007) Cell-cell and cell-extracellular matrix interactions regulate embryonic stem cell differentiation. Stem Cells 25:553–561
D'Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE (2005) Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23:1534–1541
Duan Y, Catana A, Meng Y, Yamamoto N, He S, Gupta S, Gambhir SS, Zern MA (2007) Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo. Stem Cells 25:3058–3068
Ek M, Soderdahl T, Kuppers-Munther B, Edsbagge J, Andersson TB, Bjorquist P, Cotgreave I, Jernstrom B, Ingelman-Sundberg M, Johansson I (2007) Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells. Biochem Pharmacol 74:496–503
Finta C, Zaphiropoulos P (2000) The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons. Gene 260:13–23
Fukumitsu K, Ishii T, Yasuchika K, Amagai Y, Saitoh M, Kawamoto T, Kawase E, Suemori H, Nakatsuji N, Ikai I, Uemoto S (2009) Establishment of a cell line derived from mouse fetal liver that have the character to promote the hepatic maturation of mouse embryonic stem cells by co-culture method. Tissue Eng Part A (in press)
Gellner K, Eiselt R, Hustert E, Arnold H, Koch I, Haberl M, Deglmann C, Burk O, Buntefuss D, Escher S, Bishop C, Koebe H, Brinkmann U, Klenk H, Kleine K, Meyer U, Wojnowski L (2001) Genomic organization of the human CYP3A locus: identification of a new, inducible CYP3A gene. Pharmacogenetics 11:111–121
Hasegawa K, Cowan AB, Nakatsuji N, Suemori H (2007) Efficient multicistronic expression of a transgene in human embryonic stem cells. Stem Cells 25:1707–1712
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 39:1362–1370
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 309:68–77
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 25:3252–3260
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–G321
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–G534
Lacroix D, Sonnier M, Moncion A, Cheron G, Cresteil T (1997) Expression of CYP3A in the human liver—evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth. Eur J Biochem 247:625–634
Matsunaga T, Maruyama M, Harada E, Katsuyama Y, Sugihara N, Ise H, Negishi N, Ikeda U, Ohmori S (2004) Expression and induction of CYP3As in human fetal hepatocytes. Biochem Biophys Res Commun 318:428–434
Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N (2000) Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 97:11307–11312
Stevens J (2006) New perspectives on the impact of cytochrome P450 3A expression for pediatric pharmacology. Drug Discov Today 11:440–445
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 345:926–932
Westlind A, Malmebo S, Johansson I, Otter C, Andersson T, Ingelman-Sundberg M, Oscarson M (2001) Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43. Biochem Biophys Res Commun 281:1349–1355
Wilkinson G (2005) Drug metabolism and variability among patients in drug response. N Engl J Med 352:2211–2221
Wojnowski L (2004) Genetics of the variable expression of CYP3A in humans. Ther Drug Monit 26:192–199
Acknowledgements
We thank Dr. Andrew G. Farr for providing the anti-gp38 antibodies.
Author information
Authors and Affiliations
Corresponding author
Additional information
Financial support
This work was supported in part by grants from the New Energy and Industrial Technology Development Organization (NEDO) and the Scientific Research Fund of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary Fig. 1
(JPEG 119 kb)
Rights and permissions
About this article
Cite this article
Ishii, T., Yasuchika, K., Fukumitsu, K. et al. In vitro hepatic maturation of human embryonic stem cells by using a mesenchymal cell line derived from murine fetal livers. Cell Tissue Res 339, 505–512 (2010). https://doi.org/10.1007/s00441-009-0906-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00441-009-0906-7