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ToxSci Advance Access published online on July 13, 2006
Toxicological Sciences, doi:10.1093/toxsci/kfl058
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© The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received May 19, 2006
Accepted July 5, 2006

In Vitro Toxicology

Sequential Exposure to Cytokines Reflecting Embryogenesis: The Key for In Vitro Differentiation of Adult Bone Marrow Stem Cells into Functional Hepatocyte-Like Cells

Sarah Snykers 1 *, Tamara Vanhaecke 1, Peggy Papeleu 1, Aernout Luttun 2, Yuehua Jiang 2, Yvan Vander Heyden 3, Catherine Verfaillie 2, and Vera Rogiers 1

1 Dept. Toxicology, Vrije Universiteit Brussel (VUB), B-1090 Brussels, Belgium
2 Stem Cell Institute, University of Minnesota, Minneapolis, 55455 Minnesota, USA
3 Dept. Analytical Chemistry, VUB, B-1090 Brussels, Belgium

* To whom correspondence should be addressed.
Sarah Snykers, E-mail: Sarah.Snykers{at}vub.ac.be


  Abstract

Differentiation of adult bone marrow stem cells (BMSC) into hepatocyte-like cells is commonly performed by continuous exposure to a cytokines-cocktail. Here, it is shown that the differentiation efficacy in vitro can be considerably enhanced by sequential addition of liver-specific factors [fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone (Dex)] in a time-dependent order that closely resembles the secretion pattern during in vivo liver embryogenesis.

Quantitative RT-PCR analysis and immunocytochemistry showed that, upon sequential exposure to liver-specific factors, different stages of hepatocyte differentiation, as seen during liver embryogenesis, can be mimicked. Indeed, expression of the early hepatocyte markers alpha-fetoprotein (AFP) and hepatocyte nuclear factor (HNF)3{beta} decreased as differentiation progressed, whereas levels of the late liver-specific markers albumin (ALB), cytokeratin (CK)18 and HNF1{alpha} were gradually upregulated. In contrast, cocktail-treatment did not significantly alter the expression pattern of the hepatic markers. Moreover, sequentially-exposed cells featured highly-differentiated hepatic functions, including ALB secretion, glycogen storage, urea production and inducible cytochrome P450 (CYP)-dependent activity, far more efficiently compared to the cocktail condition.

In conclusion, sequential induction of the differentiation process, analogous to in vivo liver development, is crucial for in vitro differentiation of adult rat bone marrow stem cells into functional hepatocyte-like cells. This model may not only be applicable for in vitro studies of endoderm differentiation but it also provides a ‘virtually unlimited’ source of functional hepatocytes, suitable for preclinical pharmacological research and testing, and cell and organ development.

Keywords: bone marrow stem cells; hepatocytes; sequential differentiation; liver-specific growth factors; liver embryonic development; in vitro.
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