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Embryonic stem (ES) cells provide a unique source for tissue regeneration. We examined whether mouse ES cells can efficiently differentiate into transplantable hepatocytes. ES cells were implanted into mouse livers 24 hours after carbon tetrachloride intoxication; ES-derived cells with several hepatocyte-cell-markers were generated. They were able to grow in vitro and showed morphology consistent with typical mature hepatocytes and expressed hepatocyte-specific genes. After transplantation into the carbon tetrachloride-injured mouse liver, ES-derived green fluorescent protein-positive cells were incorporated into liver tissue and rescued mice from hepatic injury. No teratoma formation was observed in the transplant recipients. In conclusion, ES cells can provide a valuable tool for studying the molecular basis for differentiation of hepatocytes and form the basis for cell therapies. (HEPATOLOGY 2003;37:983-993.)

The molecules responsible for hepatic differentiation from embryonic stem (ES) cells have yet to be elucidated. Here we have identified growth factors that allow direct hepatic fatespecification from ES cells by using simple adherent monolayer culture conditions. ES cell-derived hepatocytes showed liver-specific characteristics, including several metabolic activities, suggesting that ES cells can differentiate into functional hepatocytes without the requirement for embryoid body (EB) formation, in vivo transplantation, or a coculture system. Most importantly, transplantation of ES cell-derived hepatocytes in mice with cirrhosis showed significant therapeutic effects. In conclusion, this novel system for hepatic fate specification will help elucidate the precise molecular mechanisms of hepatic differentiation in vitro and could represent an attractive approach for developing stem cell therapies for treatment of hepatic disease in humans. M ouse embryonic stem (ES) cells are capable of differentiating into any adult animal cell type. 1,2 Although hepatocyte differentiation from mouse ES cells has been achieved 3-11 the utility of ES cells as (1) a developmental model and (2) a source for pharmaceutical screening and transplantation is hampered by limited control over the differentiation process. To address this limitation, we have established a highly reproducible in vivo method to acquire abundant functional hepatocytes from ES cells through liver-regenerating animals. 3 These hepatocytes express several differentiation markers of mature hepatocytes and showed sufficient functions to rescue experimental liver injury when transplanted in vivo. Our next goal was to elucidate the molecules responsible for directing this hepatic differentiation from ES cells, and to apply this information to developing a reproducible hepatic differentiation system in vitro.To this end, we applied the results obtained from DNA-chip analysis to induce the direct differentiation of functional hepatocytes from an adherent monolayer culture of ES cells. These cells display the characteristics of mature hepatocytes apropos of liver-specific gene expression and biochemical analysis in vitro. Importantly, transplantation of monolayer-differentiated ES cell-derived hepatocytes improved liver function and prolonged the survival of mice with cirrhosis. Although the use of human ES cells for therapeutic purposes must be rigidly controlled, the procedure described in this study for inducing transplantable hepatic cells from mouse ES cells is, theoretically, transferable to human ES cells, leading to

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The formation of the hyaluronan-rich cumulus extracellular matrix is crucial for female fertility and accompanied by a transesterification reaction in which the heavy chains (HCs) of inter-␣-trypsin inhibitor (I␣I)-related proteins are covalently transferred to hyaluronan. Tumor necrosis factor-induced protein-6 (TNFIP6) is essential for this transfer reaction. Female mice deficient in TNFIP6 are infertile due to the lack of a correctly formed cumulus matrix. In this report, we characterize the specificity of TNFIP6-mediated HC transfer from I␣I to hyaluronan. Hyaluronan oligosaccharides with eight or more monosaccharide units are potent acceptors in the HC transfer, with longer oligosaccharides being somewhat more efficient. Epimerization of the N-acetylglucosamine residues to N-acetyl-galactosamines (i.e. in chondroitin) still allows the HC transfer although at a significantly lower efficiency. Sulfation of the N-acetylgalactosamines in dermatan-4-sulfate or chondroitin-6-sulfate prevents the HC transfer. Hyaluronan oligosaccharides disperse cumulus cells from expanding cumulus cell-oocyte complexes with the same size specificity as their HC acceptor specificity. This process is accompanied by the loss of hyaluronan-linked HCs from the cumulus matrix and the appearance of oligosaccharidelinked HCs in the culture medium. Chondroitin interferes with the expansion of cumulus cell-oocyte complexes only when added with exogenous TNFIP6 before endogenous hyaluronan synthesis starts, supporting that chondroitin is a weaker HC acceptor than hyaluronan. Our data indicate that TNFIP6-mediated HC transfer to hyaluronan is a prerequisite for the correct cumulus matrix assembly and hyaluronan oligosaccharides and chondroitin interfere with this assembly by capturing the HCs of the I␣I-related proteins.In response to the luteinizing hormone (LH) 1 surge, the cumulus cell-oocyte complex (COC) in the preovulatory follicle undergoes immense changes, which results in the formation of an expanded extracellular matrix (1). This matrix is involved in the expulsion of the COC from the follicle (2), enhances the pickup and transport of the COC by the oviduct (3, 4), and influences sperm penetration and oocyte fertilization (5-7). The cellular events of matrix formation, which include the synthesis and organization of hyaluronan (HA) around the cumulus cells, have been studied extensively in mouse COCs both in vivo and in vitro (8). After the LH surge, COCs quickly upregulate the expression of hyaluronan synthase 2, the enzyme required to produce hyaluronan (9). Although the mediators that induce hyaluronan synthesis in the COCs in vivo have yet to be determined, prostaglandin E 2 , follicular stimulating hormone and epidermal growth factor are potent initiators of hyaluronan production in vitro (10 -13). Hyaluronan synthesis is also under the control of the oocyte (14, 15), and growth differentiation factor-9 and bone morphogenic protein 15 are the most likely oocyte-derived growth factors involved in this regulation (16,17).Several...