Effect of methylsulfonylmethane on oxidative stress and CYP3A93 expression in fetal horse liver cells.

OBJECTIVE: Stress-induced cytotoxicity caused by xenobiotics and endogenous metabolites induces the production of reactive oxygen species and often results in damage to cellular components such as DNA, proteins, and lipids. The cytochrome P450 (CYP) family of enzymes are most abundant in hepatocytes, where they play key roles in regulating cellular stress responses. We aimed to determine the effects of the antioxidant compound, methylsulfonylmethane (MSM), on oxidative stress response, and study the cytochrome P450 family 3 subfamily A (CYP3A) gene expression in fetal horse hepatocytes. METHODS: The expression of hepatocyte markers and CYP3A family genes (CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, and CYP3A97) were assessed in different organ tissues of the horse and fetal horse liver-derived cells (FHLCs) using quantitative reverse transcription polymerase chain reaction. To elucidate the antioxidant effects of MSM on FHLCs, cell viability, levels of oxidative markers, and gene expression of CYP3A were investigated in H2O2-induced oxidative stress in the presence and absence of MSM. RESULTS: FHLCs exhibited features of liver cells and simultaneously maintained the typical genetic characteristics of normal liver tissue; however, the expression profiles of some liver markers and CYP3A genes, except that of CYP3A93, were different. The expression of CYP3A93 specifically increased after the addition of H2O2 to the culture medium. MSM treatment reduced oxidative stress as well as the expression of CYP3A93 and heme oxygenase 1, an oxidative marker in FHLCs. CONCLUSION: MSM could reduce oxidative stress and hepatotoxicity in FHLCs by altering CYP3A93 expression and related signaling pathways.

Fetal liver cell-containing hybrid organoids improve cell viability and albumin production upon transplantation.

Cell transplantation is a potential alternative for orthotopic liver transplantation because of the chronic donor shortage. Functional liver tissue is needed for cell transplantations. However, large functional liver tissue is difficult to construct because of the high oxygen consumption of hepatocytes. In our previous study, we developed a novel method to generate hybrid organoids. In this study, we used fetal liver cells (FLCs) to construct a hybrid organoid. Nucleus numbers, angiogenesis, and albumin production were measured in transplanted samples. Higher cell viability and larger liver tissue was found in FLC-containing samples than in hepatocyte-containing samples. Furthermore, the therapeutic efficiency of FLC-containing samples was evaluated by transplantation into Nagase analbuminemia rats. As a result, an increase in albumin concentration was found in rat blood. In summary, transplantation of a FLC-containing hybrid organoid is a potential approach for cell transplantation.

Chemokine-mediated robust augmentation of liver engraftment: a novel approach.

Effective repopulation of the liver is essential for successful clinical hepatocyte transplantation. The objective was to improve repopulation of the liver with human hepatocytes using chemokines. We used flow cytometry and immunohistochemistry assays to identify commonly expressed chemokine receptors on human fetal and adult hepatocytes. The migratory capacity of the cells to various chemokines was tested. For in vivo studies, we used a nude mouse model of partial hepatectomy followed by intraparenchymal injections of chemokine ligands at various concentrations. Human fetal liver cells transformed with human telomerase reverse transcriptase were used for intrasplenic cell transplantation. Repopulation and functionality were assessed 4 weeks after transplantation. The receptor CXCR3 was commonly expressed on both fetal and adult hepatocytes. Both cell types migrated efficiently toward corresponding CXC chemokine ligands 9, 10, and 11. In vivo, animals injected with recombinant chemokines showed the highest cell engraftment compared with controls (p<.05). The engrafted cells expressed several human hepatic markers such as cytokeratin 8 and 18 and albumin as well as transferrin, UGT1A1, hepatocyte nuclear factor (1α, 1ß, and 4α), cytochrome CYP3A1, CCAAT/enhancer binding protein (α and ß), and human albumin compared with controls. No inflammatory cells were detected in the livers at 4 weeks after transplantation. The improved repopulation of transplanted cells is likely a function of the chemokines to mediate cell homing and retention in the injured liver and might be an attractive strategy to augment repopulation of transplanted hepatocytes in vivo.

Phenotypic and in vivo functional characterization of immortalized human fetal liver cells.

We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.

Engineering of human hepatic tissue with functional vascular networks.

Although absolute organ shortage highlights the needs of alternative organ sources for regenerative medicine, the generation of a three-dimensional (3D) and complex vital organ, such as well-vascularized liver, remains a challenge. To this end, tissue engineering holds great promise; however, this approach is significantly limited by the failure of early vascularization in vivo after implantation. Here, we established a stable 3D in vitro pre-vascularization platform to generate human hepatic tissue after implantation in vivo. Human fetal liver cells (hFLCs) were mixed with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) and were implanted into a collagen/fibronectin matrix composite that was used as a 3-D carrier. After a couple of days, the fluorescent HUVECs developed premature vascular networks in vitro, which were stabilized by hMSCs. The establishment of functional vessels inside the pre-vascularized constructs was proven using dextran infusion studies after implantation under a transparency cranial window. Furthermore, dynamic morphological changes during embryonic liver cell maturation were intravitaly quantified with high-resolution confocal microscope analysis. The engineered human hepatic tissue demonstrated multiple liver-specific features, both structural and functional. Our new techniques discussed here can be implemented in future clinical uses and industrial uses, such as drug testing.

Immunology research on human fetal liver cells transplantation in treatment of metabolic diseases / 上海第二医科大学学报

Objective To investigate the immunological reaction,serum and liver ?-L-iduronidase(IDUA) activity after human fetal liver cells(FLCs) transplantation therapy on mucopolysaccharidosis(MPS) which attribute to IDUA defect.Methods FLCs were transplanted into IDUA deficiency mice.T cell subsets(CD3,CD4 and CD8) was detected by flow cytometry,while IL-2,TNF-? and IFN-? by ELISA,and serum and liver IDUA activity by fluorospectrophotometer at different stage(before transplantation and 5,10,15 days after transplantation).Results Human FLCs survived in IDUA deficiency mice,bringing elevated serum and liver enzyme activity to receptor.T cell subsets,IL-2,TNF-? and IFN-? was significantly higher 5,10 and 15 days after transplantation than those before transplantation(P

THERAPEUTIC EFFECT OF INTRAMEDULLARY INJECTION OF FETAL LIVER CELL SUSPENSION ON THE LETHALLY IRRADIATED MICE / 第二军医大学学报

This paper describes the study on intramedullary injection of fetal liver cell suspension into the lethally irradiated mice. The results show that the therapeutic effect of intramedullary injection is better than that of intravenous one.It is suggested that the fetal liver cells injected into the bone marrow cavity can settle down and proliferate here and the volume of injection should be limited.

HOMOLOGOUS FETER LIVER CELLS FOR SEVERE VIRAL HEPATITIS: CONTROLLED TRIALS ON 30 CASES / 解放军医学杂志

Fifteen patients with hepatocellular failure from viral hepatitis were treated with general combined therapy and transfusion" of homologous fetal hepatic cells (FLO for 36 times (group A). An additional 15 cases with hepatocellular failure from viral hepatitis were treated only with general combined therapy, as control (group B). Twelve cases of the group A were diagnosed by transcutaneous liver biopsy.The results showed that FLC suspension achieved satisfactory effects and those who received FLC rallied remarkably better than those who did not. The fatality rates of group A and B were 26.7% (4/15) and 60.0% (9/15) respectively. In group A after transfusion of FLC, the effects were mainly shown by the elevation of the prothrombin activity (elevated for 16.5% after 24 h and for 18.2% after 48 h in average), recovery from hepatic coma and improvement in the subjective symptoms and various hepatic function tests. In survivals of group A (11 cases), the serum bilirubin returned to normal more quickly than that of group B (5 cases), being 55.4?24.5 days and 119.7?54.9 days, respectively (P