There is now abundant evidence to substantiate an important role of hepatitis C virus (HCV) core protein in cellular gene expression as well as in the viral cycle. Thus the subcellular localization of this protein has important implications. However, several studies have shown controversial results: the HCV core has been, indeed, described as cytoplasmic or nuclear depending on the size of the protein or on the genotype analyzed. We have studied the localization of the HCV core protein in two different cell lines, one nonhepatic (CHO) and the other hepatic (HepG2). Double immunofluorescence staining using a nuclear membrane marker and confocal analysis showed the core protein pattern to be cytoplasmic and globular. This pattern is not cell cycle-regulated. Electron microscopy analysis revealed the nature of the globular staining observed in immunofluorescence. The HCV core protein accumulated at the surface of lipid droplets that were also the unique morphological feature of nonhepatic core transfected cells. The lipid droplets were isolated by sequential ultracentrifugation on the basis of their density; biochemical analysis revealed a prevalence of triglycerides. In addition the core protein colocalized with apolipoprotein AII at the surface of the lipid droplets as revealed by confocal microscopy. Moreover analysis of liver biopsies from chronically HCV-infected chimpanzees revealed that HCV core is cytoplasmic and localized on the endoplasmic reticulum and on lipid droplets. These results clearly define the subcellular localization of the HCV core protein and suggest a relationship between the expression of the HCV core protein and cellular lipid metabolism.

Epidemiological data indicate that dietary tocopherols and carotenoids can exert cardioprotective effects, which may be mediated by their antioxidant actions. The oxidative modification of LDL underlies the atherogenicity of these cholesterol-rich particles. The resistance of LDL to oxidation is influenced by several endogenous factors, among which the content of tocopherols and carotenoids is prominent. Of the exogenous factors, HDL inhibits oxidation of LDL via several mechanisms. In view of the paucity of data on the distribution of diverse tocopherol and carotenoid components among the apoB- and apoA-I–containing lipoproteins of human plasma, we evaluated the quantitative and qualitative features of the LDL and HDL particle subspecies in normolipidemic subjects. The bulk of tocopherols and hydrocarbon carotenoids (lycopene, α- and β-carotene) was transported in LDL (45% and 76%, respectively), in contrast to the oxygenated carotenoids (lutein/zeaxanthin, canthaxanthin, and β-cryptoxanthin), which were equally distributed between LDL and HDL. α-Tocopherol content was independently associated with lipid core size (cholesteryl ester and triglyceride) in VLDL, LDL, and HDL (P<.005); by contrast, the particle content of the oxygenated carotenoids lutein/zeaxanthin and canthaxanthin was strongly related to that of phospholipids. A significant and progressive decrease in the molar content of α- and γ-tocopherols was found with increase in density from light to dense LDL subspecies (LDL1 to LDL5); a similar trend was observed in HDL subspecies. Furthermore, particle contents of lutein/zeaxanthin, β-cryptoxanthin, β-carotene, and lycopene were markedly reduced in small, dense LDL (LDL5,d=1.050 to 1.065 g/mL). We conclude that diminished contents in such carotenoids as well as in tocopherols could underlie not only the diminished oxidative resistance of small, dense LDL but also reduced tissue targeting of antioxidants in subjects with a dense LDL phenotype.

The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA-based or virus-based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA-derived iPSCs with that of retrovirus-derived iPSCs generated in strictly comparable conditions, by single-nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA-derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus-derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone-dependent. Furthermore, differentiation studies indicated that mRNA-derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration-free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell-derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications.

Background: Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods.Results: We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration.Conclusions: We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also forin vitroapplications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC) decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL‐MSCs). After isolation from 11‐12 gestational weeks’ human fetal livers, FL‐MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblastic differentiation potential. Thereafter, we explored their hepatocytic differentiation potential using the hepatogenic protocol applied for adult human liver mesenchymal cells. FL‐MSCs differentiated in this way displayed significant features of hepatocyte‐like cells as demonstrated in vitro by the upregulated expression of specific hepatocytic markers and the induction of metabolic functions including CYP3A4 activity, indocyanine green uptake/release, and glucose 6‐phosphatase activity. Following transplantation, naive and differentiated FL‐MSC were engrafted into the hepatic parenchyma of newborn immunodeficient mice and differentiated in situ. Hence, FL‐MSCs appeared to be interesting candidates to investigate the liver development at the mesenchymal compartment level. Standardization of their isolation, expansion, and differentiation may also support their use for liver cell‐based therapy development.

1. The pharmacokinetics of the mucolytic compound ³⁵S-cysteine ethyl ester in rat show that it is completely absorbed after oral administration, with a bioavailability of 0˙59. 2. By autoradiography, tissue distribution of ³⁵S-cysteine ethyl ester showed a high affinity for the lung, different to that observed with ³⁵S-cysteine. 3. Unchanged cysteine ethyl ester, cysteine and inorganic sulphates were present in the lung. 4. After oral or i.v. administration of ³⁵S-cysteine ethyl ester the total radioactivity excreted in the urine amounted to 16% dose and 40% was excreted in the faeces, following biliary excretion. It was metabolized to inorganic sulphate, cysteine and taurine, which were excreted in the urine.