Cellular and Molecular Biology of the Hepatic Stem Cell Compartment

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We have established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP positive cells expressing ALB were detected in close proximity to beating myocytes after 7 days of EB cultures. GFP positive cells increased in number, acquired hepatocyte-like morphology and hepatocyte specific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl4-induced liver injury. The transplanted GFP positive cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy. The 2-acetaminofluorene/partial hepatectomy (AAF/PH) model is widely used to induce oval/progenitor cell proliferation in the rat liver. We have used this model to study the impact of a primary hepatocyte mitogen, triiodothyronine (T3) on the liver regenerating by the recruitment of oval/progenitor cells. Administration of triiodothyronine (T3) on the 5th day after the PH can not induce mitogenic reaction in the hepatocytes. However, the oval cells respond by accelerated proliferation, which is followed by rapid differentiation into small hepatocytes. During the accelerated differentiation the oval cell specific OV-6 and alpha-fetoprotein (AFP) expression is lost, hepatocyte nuclear factor (HNF)-4 is upregulated in newly formed small, basophilic hepatocytes. The differentiating oval cells also start to express hepatocyte specific connexin (Cx) 32, 1 integrin and cytochrom P450s, and form CD 26 positive bile canaliculi. The upregulation of hepatocyte specific mRNAs e.g. albumin, , tyrosine aminotransferase ( TAT) and tryptophan 2,3-dioxygenase (TO2) was detected by by quantitative real time PCR in microdissected small hepatocytes. Furthermore, improved liver function tests happens concurrently with the accelerated differentiation. The hepatocytic conversion of oval cells occurs on the 7th day after the Phx in this model while the first small hepatocytes appear on the 11th-12th day post Phx without T3 treatment. We conclude that administration of the primary hepatocyte mitogen T3 accelerates the differentiation of hepatic progenitor cells into hepatocytes in vivo, and that may have therapeutic potential. The HGF/Met signaling system is essential for liver development, homeostasis and function. In this study, we took advantage of the liver specific Met conditional knockout mouse generated in our laboratory previously to address the role of Met in adult liver progenitor cell (oval cell) biology. For this purpose, we isolated oval cells from DDC-treated Metflx/flx mice, and then established oval cell derived cell lines that carried either functional (Metflx/flx) or non-functional (Met-/-) met gene using virus-mediated Cre-loxP recombination. Oval cells lacking Met kinase activity were phenotypically similar to Metflx/flx controls but displayed neither Met phosphorylation, nor activation of Met downstream targets. In addition, Met-/- cells were refractory to HGF stimulation of proliferation. Although Met-/- and Metflx/flx cells proliferated at a similar rate in the presence of 10% serum, Met deficient cells demonstrated decreased cell viability, and were more prone to apoptosis when challenged with serum starvation or the pro-apoptotic cytokine TGF-beta. Treatment with HGF reduced the TGF-Beta-mediated cell death in Metflx/flx but not Met-/- cells. Importantly, both Metflx/flx and Met-/- cells constitutively expressed HGF, and conditioned medium from serum starved oval cells exhibited antiapoptotic activity in Metflx/flx cells. Furthermore, serum-starved Metflx/flx cells showed persistent activation of Met tyrosine kinase suggesting HGF/Met autocrine regulation. In conclusion, these data assign a critical functional role for Met in oval cell survival through an autocrine mechanism