Embryoid‐body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes

Embryonic stem (ES) cells have a potential to differentiate into various progenitor cells. Here we investigated the differentiation capacity of mouse ES cells into hepatocytes both in vitro and in vivo. During the culture of embryoid bodies (EBs) derived from ES cells, albumin (ALB) messenger RNA (mRNA) was expressed within 12 days after removal of leukemia inhibitory factor, and α‐fetoprotein (AFP) mRNA was observed within 9 days without additional exogenous growth factors. In ES cells and early EBs, by contrast, neither ALB mRNA nor AFP mRNA was observed. ALB protein was first detected at day 15 and the level increased with the culture period. The differentiation of EBs facilitated the synthesis of urea with the culture period, whereas early EBs and ES cells produced no urea. These results suggest that cultured EBs contain hepatocytes capable of producing ALB and urea. ES cells and the isolated cells from EBs were transplanted through portal vein to the liver after 30% partial hepatectomy of female mice pretreated with 2‐acetylaminofluorene. Four weeks after transplantation with isolated cells from day‐9 EBs, ES‐derived cells containing Y‐chromosome in the liver were positive for ALB (0.2% of total liver cells), whereas teratoma was found in mice transplanted with ES cells or EBs up to day 6. The incidence of teratoma was decreased with the culture duration and no teratoma was observed in the liver transplanted with isolated cells from day‐9 EBs. In conclusion, our in vitro and in vivo experiments revealed that cultured EBs contain functional hepatocytes or hepatocyte‐like cells. (HEPATOLOGY 2002;36:22‐29.)

[1]  Yoshiaki Ito,et al.  Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer , 1999, The EMBO journal.

[2]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[3]  G. Schütz,et al.  Hepatocyte differentiation in vitro: initiation of tyrosine aminotransferase expression in cultured fetal rat hepatocytes , 1989, The Journal of cell biology.

[4]  M. Frain,et al.  Differential expression of albumin and α-fetoprotein genes in fetal tissues of mouse and rat , 1984 .

[5]  M. Wiles,et al.  Hematopoietic commitment during embryonic stem cell differentiation in culture. , 1993, Molecular and cellular biology.

[6]  M. Kaufman,et al.  Establishment in culture of pluripotential cells from mouse embryos , 1981, Nature.

[7]  W. H. Marsh,et al.  AUTOMATED AND MANUAL DIRECT METHODS FOR THE DETERMINATION OF BLOOD UREA. , 1965, Clinical chemistry.

[8]  W. Schulz,et al.  Coexpression of the C-MYC protooncogene with α-fetoprotein and albumin in fetal mouse liver , 1990 .

[9]  K. Burton [105] Determination of DNA concentration with diphenylamine , 1968 .

[10]  K. Mizuseki,et al.  Induction of Midbrain Dopaminergic Neurons from ES Cells by Stromal Cell–Derived Inducing Activity , 2000, Neuron.

[11]  D. Gottlieb,et al.  Embryonic stem cells express neuronal properties in vitro. , 1995, Developmental biology.

[12]  R Kemler,et al.  The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. , 1985, Journal of embryology and experimental morphology.

[13]  J. Chou Regulators of fetal liver differentiation in vitro. , 1988, Archives of biochemistry and biophysics.

[14]  D. Friend,et al.  HIGH-YIELD PREPARATION OF ISOLATED RAT LIVER PARENCHYMAL CELLS , 1969, The Journal of cell biology.

[15]  Naohiro Terada,et al.  Hepatic maturation in differentiating embryonic stem cells in vitro , 2001, FEBS letters.

[16]  Donald Metcalf,et al.  Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells , 1988, Nature.

[17]  G. Martin,et al.  Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[18]  John K. Heath,et al.  Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides , 1988, Nature.

[19]  R. McKay,et al.  Differentiation of Embryonic Stem Cells to Insulin-Secreting Structures Similar to Pancreatic Islets , 2001, Science.

[20]  P. Leder,et al.  Leukemia inhibitory factor is expressed by the preimplantation uterus and selectively blocks primitive ectoderm formation in vitro. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[21]  D. Melton,et al.  Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. Bernuau,et al.  Fulminant and Subfulminant Liver Failure: Definitions and Causes , 1986, Seminars in liver disease.

[23]  K. Zaret,et al.  Initiation of mammalian liver development from endoderm by fibroblast growth factors. , 1999, Science.

[24]  H. Snodgrass,et al.  Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. , 1991, Genes & development.

[25]  Tetsuo Noda,et al.  Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor , 1995, Nature.

[26]  M. Sakaguchi,et al.  Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro. , 2000, Biochemical and biophysical research communications.

[27]  A. Trounson,et al.  Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro , 2000, Nature Biotechnology.

[28]  L. Samuelson,et al.  Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells , 1994, The Journal of cell biology.

[29]  Rebekah Gardner,et al.  Liver from bone marrow in humans , 2000, Hepatology.

[30]  M. Sharpe,et al.  Scatter factor/hepatocyte growth factor is essential for liver development , 1995, Nature.

[31]  T. Doetschman,et al.  Myosin heavy chain gene expression in mouse embryoid bodies. An in vitro developmental study. , 1991, The Journal of biological chemistry.