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Differentiation of human Embryonic Stem Cells into hepatocytes as a tool to analyse dynamic regulatory events during hepatogenesis in vitro.


Józefczuk,  Justyna
Max Planck Society;

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Józefczuk, J. (2009). Differentiation of human Embryonic Stem Cells into hepatocytes as a tool to analyse dynamic regulatory events during hepatogenesis in vitro. PhD Thesis, Freie Universität Berlin, Berlin.

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The differentiation of human embryonic stem cells (hESCs) into functional hepatocytes provides a powerful in vitro model system for analysing the molecular mechanisms involved in liver development. Moreover, a well-characterized renewable source of hepatocytes differentiated from human ES cells could be used for in vitro assays of drug metabolism and toxicology. The mechanisms underlying the molecular embryological events leading to fetal liver development has been studied by genetic analysis in rodents and explants studies in the model organisms such as the chick embryo. Nowadays the pluripotent characteristics of human ES cells provide great promise for the study of early development in man. Especially because protocols leading to differentiation of human ES cells into cells possessing morphologic and molecular features typical for hepatocytes are available. Genetic studies in the model organisms have unveiled the major inducers of hepatogenesis such as FGF4, BMP2, HGF, oncostatin M and dexamethasone. Currently the biggest challenge is to gain insight into target genes and associated signaling pathways regulated by extracellular ligands, which are normally sequentially supplemented into in vitro differentiation protocols. This knowledge will in turn enhance our understanding of the dynamic developmental events at the transcript and proteome level. This study was aimed at elucidating mechanisms of in vitro hepatogenesis, which mimics the in vivo process. In order to performed time-resolved analysis of the process of hepatogenesis, I used a reproducible in vitro system (two different protocols) and analysed the transcriptomes of the differentiating human ES cells in a step-wise manner (definitive endoderm induction, early hepatic cells, hepatic maturation) throughout the entire process. This in vitro analysis has revealed a broad spectrum of molecular cascades involving cell surface receptors, transcriptional regulators and associated signaling pathways that might be driving hepatogenesis in vivo. In addition, these experiments revealed that at the transcriptional level human ES cells-derived hepatocyte-like cells have some similarity to fetal liver, but in some aspects present an individual, exclusive transcription pattern. In summary, the results suggest that an in vitro system for hepatic differentiation is a potent tool for analyzing molecular pathways involved at each stage of this process. However, further effort is required to obtain more mature cells, and the knowledge gained from the transcript analysis might aid in achieving this goal.