Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury-induced donor hepatocyte selection: the uPA/SCID mice and Fab(-/-) Rag2(-/-) II2rg(-/-) mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GMI and FK506 in Fab(-/-) Rag2(-/-) mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fab(-/-) Rag2(-/-) mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and FIBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharma- cokinetics and efficacy of potential vaccines. (Am J Pathol 2010. 177:1311-1319; 10.2353/aypath.2010.091154)

Stable epigenetic silencing of p16(INK4a) is a common event in hepatocellular carcinoma (HCC) cells, which is associated with abnormal cell proliferation and liberation from cell cycle arrest. Understanding the early epigenetic events in silencing p16(INK4a) expression may illuminate a prognostic strategy to block HCC development. Toward this end, we created a reprogram cell model by the fusion mouse HCC cells with mouse embryonic stem cells, in which the ES-Hepa hybrids forfeited HCC cell characteristics along with reactivation of the silenced p16(INK4a). HCC characteristics, in terms of gene expression pattern and tumorigenic potential, was restored upon induced differentiation of these reprogrammed ES-Hepa hybrids. The histone methylation pattern relative to p16(INK4a) silencing during differentiation of the ES-Hepa hybrids was analyzed. H3K27 trimethylation at the p16(INK4a) promoter region, occurring in the early onset of p16(INK4a) silencing, was followed by H3K9 dimethylation at later stages. During the induced differentiation of the ES-Hepa hybrids, H3K4 di- and trimethylations were maintained at high levels during the silencing of p16(INK4a), strongly suggesting that H3K4 methylation events did not cause the silencing of p16(INK4a). Our results suggested that the enrichment of H3K27 trimethylation, independent of H3K9 dimethylation, trimethylation, and DNA methylation, was an early event in the silencing of p16(INK4a) during the tumor development. This unique chromatin pattern may be a heritable marker of epigenetic regulation for p16(INK4a) silencing during the developmental process of hepatocellular carcinogenesis.

Induction of definitive endoderm (DE) cells is a prerequisite for the whole process of embryonic stem (ES) cells differentiating into hepatic or pancreatic progenitor cells. We have established an efficient method to induce mouse ES cell-derived DE cells in suspension embryonic body (EB) culture. Similar to previous studies, mouse ES cell-derived DE cells, which were defined as Cxcr4(+)c-Kit(+),Cxcr4(+)E-cadherin(+) cells or Cxcr4(+)PDGFRa(-) cells, could be induced in the serum-free EBs at Day 4 of induction. The activations of Wnt, Nodal, and FGF signaling pathways in differentiating EBs promoted DE cell differentiation, while activation of BMP4 signaling inhibited the process. In the present study, we found that chemical activation of canonical Wnt signaling pathway by LiCl could synergize with Activin A-mediated Nodal signaling pathway to promote induction of DE cells, and inhibition of Bmp4 signaling by Noggin along with Activin A/LiCl further improved the efficiency of DE cell differentiation. The derived DE cells were proved for their capacities to become hepatic progenitor cells or pancreatic progenitor cells. In conclusion, we significantly improved the efficiency of generating mouse ES cell-derived DE cells by combined Activin A/LiCl/Noggin treatment. Our work will be greatly helpful to generate ES cell-derived hepatic cells and ES cell-derived pancreatic cells for future regenerative medicine. J. Cell. Biochem. 112: 1022-1034, 2011. (C) 2010 Wiley-Liss, Inc.

Although embryonic stem (ES) cell-derived hepatocytes have the capacity for liver engraftment and repopulation, their in vivo hepatic function has not been analyzed yet. We aimed to determine the metabolic function and therapeutic action of ES cell-derived hepatocytes after serial liver repopulations in fumaryl acetoacetate hydrolase knockout (Fah(-/-)) mice. Albumin expressing (Alb(+)) cells were obtained by hepatic differentiation of ES cells using two frequently reported methods. After transplantation, variable levels of liver repopulation were found in Fah(-/-) mice recipients. FAH expressing (FAH) hepatocytes were found either as single cells or as nodules with multiple hepatocytes. After serial transplantation, the proportion of the liver that was repopulated by the re-transplanted FAH(+) hepatocytes increased significantly. ES cell-derived FAH. hepatocytes were found in homogenous nodules and corrected the liver metabolic disorder of Fah(-/-) recipients and rescued them from death. ES cell-derived hepatocytes had normal karyotype, hepatocytic morphology and metabolic function both in vitro and in vivo. In conclusion. ES cell-derived hepatocytes were capable of liver repopulation and correction of metabolic defects after serial transplantation. Our results are an important piece of evidence to support future clinical applications of ES cell-derived hepatocytes in treating liver diseases. (C) 2012 Elsevier Ltd. All rights reserved.

发展有效的抗纤维化方案在临床肝脏疾病治疗中具有重要意义。细胞外基质(ECM)沉积是肝纤维化发生发展的主要病理基础,ECM的过度沉积与基质金属蛋白酶家族(MMPs)和金属蛋白酶组织抑制因子(TIMPs)表达的平衡失调密切相关。通过调节MMPs/TIMPs的活性，促进已有基质的吸收，是治疗肝纤维化的有效策略。为了解决病毒载体应用的安全性和非靶向表达等问题，本课题应用新型SB转座子构建MMPs/TIMPs非病毒表达载体，将其导入Fah基因剔除小鼠肝纤维化模型，靶向调控MMPs/TIMPs在肝细胞中的表达活性，克服了病毒载体的缺陷和传统非病毒载体转染效率低的难题。同时，通过IVIS系统、RT-PCR、免疫组化、组织学、血清生化指标及肝功能的检测，本课题评价了靶向调控MMPs/TIMPs表达对肝纤维化的治疗作用。本课题的研究表明，通过调控肝细胞中MMPs/TIMPs的表达能有效缓解肝纤维化，其中MMP13 SB载体具有更好的应用价值。通过本课题的实施，还为MMPs/TIMPs的临床应用建立了可行的技术体系。

BACKGROUND & AIMS: Hepatocyte-like cells can be derived from pluripotent stem cells such as embryonic stem (ES) cells, but ES cell-derived hepatic cells with extensive capacity to repopulate liver have not been identified. We aimed to identify and purify ES cell-derived hepatoblast-like progenitor cells and to explore their capacity for liver repopulation in mice after in vitro expansion. METHODS: Unmanipulated mouse ES cells were cultured under defined conditions and allowed to undergo stepwise hepatic differentiation. The derived hepatic cells were examined by morphologic, fluorescence-activated cell sorting, gene expression, and clonal expansion analyses. The capacities of ES cell-derived hepatic progenitor cells to repopulate liver were investigated in mice that were deficient in fumarylacetoacetate hydrolase (Fah) (a model of liver injury). RESULTS: Mouse ES cells were induced to differentiate into a population that contained hepatic progenitor cells; this population included cells that expressed epithelial cell adhesion molecule (EpCAM) but did not express c-Kit. Clonal hepatic progenitors that arose from single c-Kit(-)EpCAM(+) cells could undergo long-term expansion and maintain hepatoblast-like characteristics. Enriched c-Kit(-)EpCAM(+) cells and clonally expanded hepatic progenitor cells repopulated the livers of Fah-deficient mice without inducing tumorigenesis. CONCLUSIONS: ES cell-derived c-Kit(-)EpCAM(+) cells contain a population of hepatoblast-like progenitor cells that can repopulate livers of mice.