OBJECTIVETo investigate the characteristics of phase II metabolic enzymes in mouse embryonic stem (ES) cell-derived liver tissue.

METHODSMature hepatocytes were differentiated from embryonic stem cells in cultured mouse embryoid bodies (EB) at d18. Western blot was used to detect the expression of uridine 5'-diphosphate glucronosyl transferase (UGT1a1,UGT1a6) and microsomal glutathione S-transferases 1(mGST1) during the differentiation course.The derived liver tissue was incubated with UDPGA and 7-HFC,the formation of 7-HFC glucuronide was detected by HPLC to examine the total activities of UGT1a1 and UGT1a6. Furthermore, the microsomes were incubated with CDNB and GSH,and the mGST1 activity was measured by spectrometry.

RESULTSAn increase tendency of UGT1a1 expression was noticed during the differentiation course. UGT1a6 and mGST1 were not detected in the earlier stage until d18 of differentiation. The metabolic activity of mGST1 in the derived hepatocytes was 7.65 nmol/min/mg on d18.

CONCLUSIONThe ES cell-derived liver tissue possesses partial metabolic function of phase II enzymes on d18 of differentiation,which might be used as a model for in vitro research on hepatic pathophysiology and phase II drug metabolism.

Animals ; Cell Differentiation ; Embryoid Bodies ; cytology ; Embryonic Stem Cells ; cytology ; Glucuronosyltransferase ; physiology ; Glutathione Transferase ; physiology ; Hepatocytes ; cytology ; enzymology ; Mice

Animals ; Blotting, Western ; Cell Differentiation ; drug effects ; Embryonic Stem Cells ; cytology ; enzymology ; Isoenzymes ; analysis ; Mice ; Neurons ; enzymology ; Protein Kinase C ; analysis ; Tretinoin ; pharmacology

OBJECTIVETo elucidate gene expressions of phase II enzymes in the mouse embryonic stem (ES) cell-derived hepatocytes.

METHODSEmbryoid body (EB) formation cultures were applied in directed differentiation of ES to hepatic-like cells. The expressions of hepatic-specific genes, including AFP, ALB, Cyp7a1, were detected by RT-PCR during the differentiation course. Albumin was detected by immunocyto- chemistry. The gene expressions of mGST1 and UGTs family, including UGT1a1, UGT1a6, UGT1a9 and UGT2b5, were investigated using RT-PCR.

RESULTSA notable gene expression of AFP and ALB was observed on d 8. On d 18, AFP gene failed to express, while ALB and Cyp7a1 genes were detected.Albumin-positive staining was detected in hepatic-like cells. Phase II enzyme genes expressed in variance during the differentiation course, UGT1a1 and UGT1a9 were expressed stably, UGT1a6 expression increased gradually, and UGT2b5 failed to express. Little mGST1 gene expression could been detected in the early course until d 18. In addition, all the enzymes gene expressions in the derived hepatocytes on d 18 were similar to those from mature mouse hepatocytes.

CONCLUSIONSMouse ES cell-derived mature hepatocytes express phase II enzyme UGTs and mGST1 genes similar to those in mature hepatocytes. The system may offer an alternative animal testing model related to phase enzymes in further research.

Animals ; Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; enzymology ; Fluorescent Antibody Technique ; Glucuronosyltransferase ; genetics ; metabolism ; Glutathione S-Transferase pi ; genetics ; metabolism ; Hepatocytes ; cytology ; enzymology ; Isoenzymes ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Microsomes ; enzymology ; Reverse Transcriptase Polymerase Chain Reaction

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.
Biological Markers/metabolism ; Cell Differentiation/*drug effects ; Cell Line ; Cell Lineage/*drug effects ; Cells, Cultured ; Down-Regulation/drug effects ; Embryo, Mammalian/cytology/drug effects/metabolism ; Embryonic Stem Cells/*cytology/*drug effects/enzymology ; Endoderm/*cytology/drug effects ; Enzyme Activation/drug effects ; Free Radical Scavengers/pharmacology ; Humans ; Mesoderm/*cytology/drug effects ; Mitogen-Activated Protein Kinases/metabolism ; Pluripotent Stem Cells/cytology/metabolism ; Reactive Oxygen Species/metabolism/*pharmacology ; Up-Regulation/drug effects

Human embryonic stem cells (hESCs) are considered to be able to stably maintain their characteristics in vitro for prolonged periods, but we had previously encountered changes in proliferative ability and differentiation potential during extended culture of hESCs. Therefore, we investigated the proliferative ability and differentiation potential of hESCs during long-term culture. The hESCs, SNUhES3, were used to analyze population-doubling time, proliferation rate and differentiation potential. We classified hESCs into three groups according to culture period. Ten colonies of hESCs for each group were daily measured colony area and population-doubling time was assessed by the changes of colony area. Proliferation rate of hESCs was measured by 5-bromo-2'-deoxyuridine (BrdU) assay and telomerase activity. To evaluate differentiation potentials for hESCs, expression levels of undifferentiated and/or differentiated hESCs markers were examined by FACS, RT-PCR and immunostaining. Population-doubling time of early passage hESCs was longer than those of middle or late passage. Proliferative ability of hESCs was accelerated depending on culture periods. Cellular morphologies and the expression level of each three germ layer markers were obviously different from each passage of reattached embryoid bodies (EBs) after spontaneous differentiation. Differentiated cells of late passage expressed higher levels of undifferentiated markers such as Oct4 and SSEA4 than those of early and middle passage. But differentiated cells of early and middle passage expressed higher level of differentiated state markers, Nestin (ectoderm), Brachyury (mesoderm), HNF3beta (endoderm). From these results, it can be inferred that hESCs show higher proliferative abilities and reduced differentiation potentials as the passage number increased. Therefore, we conclude that early passage hESCs could be more suitable than middle and late passage hESCs in differentiation studies.
Biological Markers/metabolism ; Bromodeoxyuridine/metabolism ; *Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cyclins/metabolism ; DNA-Binding Proteins/genetics/metabolism ; Embryonic Stem Cells/*cytology/enzymology ; Flow Cytometry ; Gene Expression Regulation ; Homeodomain Proteins/genetics/metabolism ; Humans ; Karyotyping ; Octamer Transcription Factor-3/genetics/metabolism ; Telomerase/metabolism ; Time Factors