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Soto-Gutierrez A, Kobayashi N, Rivas-Carrillo JD, Navarro-Alvarez N, Zhao D, Okitsu T, Noguchi H, Basma H, Tabata Y, Chen Y, Tanaka K, Narushima M, Miki A, Ueda T, Jun HS, Yoon JW, Lebkowski J, Tanaka N, Fox IJ. Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes. Nat Biotechnol 2006;24:1412-1419. (Reprinted by permission.)

Abstract

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Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter-based cell sorting. ES cell-derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell-derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell-derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.

Comment

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Many acute and chronic end-stage liver diseases are life-threatening and require orthotopic liver transplantation as the only definitive therapeutic option. To overcome donor organ shortage, liver cell replacement strategies are a desired alternative. Primary hepatocytes, which would be the most physiologically appropriate cell source, are restricted in their timely availability and quantity. Thus, great efforts are underway to explore new strategies to substitute functional parenchymal liver cells.1 Directed differentiation of embryonic stem (ES) cells would be a welcome approach to theoretically provide unlimited numbers of metabolically active and immunologically inert cells with hepatocellular function.

In a recent study, Soto-Gutierrez et al.2 elegantly combined recent efforts to generate functional hepatocytes from mouse ES cells and applied them in a model of acute liver failure. Their differentiation protocol is simple, uses defined reagents and yields to date the most efficiently differentiated hepatocyte-like cells (Fig. 1). Starting with a suspension culture system, where early endodermal development is initiated,3 ES cells are subsequently transferred to plates and cultured in the presence of fibroblast growth factor-2 and activin A. The predifferentiated cells are then further developed toward hepatocytes in a defined coculture together with human nonparenchymal liver cells under serum-free conditions and the influence of hepatocyte growth factor, dimethyl sulfoxide, and dexamethasone. The final step in their protocol involves flow-sorting and the further use of only the most hepatocyte-reminiscent cell population. Technically, this was accomplished by selecting cells expressing green fluorescent protein (GFP) under control of the albumin promoter, thus ensuring the yield of a pure and defined population of hepatocellular differentiated cells.

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Figure 1. Procedure for differentiating functional hepatocytes from mouse ES cells. Abbreviations: FGF, fibroblast growth factor; DMSO, dimethyl sulfoxide; HGF, hepatocyte growth factor; Dex, dexamethasone; huNPL, human nonparenchymal liver cells. BAL, bioartificial liver system.

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Those ES cell–derived neo-hepatocytes have then further been shown to display an impressive metabolic capacity (70% albumin production, 49% ammonia turnover) and a similar gene expression profile compared to primary mouse hepatocytes. To prove their biological potential and functional efficiency, the cells were packaged into a miniature bioartificial liver system (BAL) and implanted into 90% partial hepatectomized mice. Mice treated in such a manner usually die without further metabolic support from cotransplanted hepatocytes. However, 90% of the mouse experimental group, which was supported by BALs loaded with ES cell–derived neo-hepatocyte (using 5 × 106 cells equivalent to 5% of the mouse liver mass), survived. All mice treated with BALs charged with undifferentiated ES cells or albumin-GFP–negative cells died from acute liver failure. Although the long-term in vivo fate of the hepatocellular-differentiated ES cells is uncertain, mice that underwent transplantation gained enough time necessary to complete liver regeneration.

In the study by Soto-Gutierrez et al., hepatocellular-differentiated murine ES cells have been impressively proven to serve as a functional liver cell replacement. Yet many questions remain to be further elucidated before such a protocol can be succesfully applied to human ES cells. One of the most particular aspects of ES cell differentiation is the question whether the cells are really differentiated in the desired way. The expression of tissue-specific markers alone is not yet a true hallmark of succesful differentiation. Cellular gene expression is very tightly regulated by the concerted action of transcription factors and other regulatory mechanisms.4 However, if those control entities are not effective enough, especially ES cells are either fated to de-differentiate or to unleash their inherent teratogenic potential. Those unwanted developments are even more a concern and thus harder to control if increased numbers of ES cells are to be considered for potential human use. Nevertheless, this work together with recent other studies5, 6 nurture the hope that stem cells will find their niche in regenerative medicine toward future applications in clinical hepatology.

References

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