Differentiation of Mouse Embryonic Stem Cells Along a Hepatocyte Lineage

Differentiation of Mouse Embryonic Stem Cells Along a Hepatocyte Lineage
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Published on 2007 by ProQuest

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The development of implantable engineered liver tissue constructs, ex vivo hepatocyte based therapeutic devices and drug discovery studies are limited by an inadequate hepatocyte cell source. Embryonic stem (ES) cells, characterized by their self-renewing and multi-lineage differentiating capabilities, represent a promising mature cell source required for these applications. Previous research has utilized embryoid body (EB) formation in both guided, through extracellular matrix and growth factor supplementation, and unguided, or spontaneous, differentiation to generate hepatocyte like cells. However, these characterizations have been limited to only one or several lineage specific protein or gene expression patterns. In addition, there have been few reports of long term propagation or characterization of long term function for ES cell derived hepatocyte precursors. In this thesis, we have implemented a platform for the long term propagation and augmentation of functional hepatocytes generated from murine ES cell sources. We first utilize a controlled, reproducible, EB mediated differentiation system to characterize efficiency of hepatocyte lineage commitment in four parallel culture configurations. These studies have shown that, EB mediated stem cell differentiation spontaneously yield populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB) and Cytokeratin 18 (CK-18). We then used secondary culture configurations to study the effects of collagen sandwich culture and Oncostatin-M (OSM) or S-nitroso- N-acetylpenicillamine (SNAP) supplementation of EB derived hepatocyte-lineage cell function. The results of these studies suggest that SNAP, independent of the collagen supplementation, maintains the highest levels of ALB expression, however mature liver specific CK-18 is only expressed in the presence of both gel sandwich culture supplemented with SNAP. In addition, albumin secretion and Cytochrome P450 detoxification studies indicated that this condition was the best for the augmentation of hepatocyte-like function. Maintenance and augmentation of hepatocyte-like cells isolated from heterogeneous EB cell populations will be a critical step in generating large numbers of functional differentiated cells for therapeutic use.

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