title: Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.
abstract: We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.
authors.affiliation: Department of Pharmacy, Shinshu University Hospital, 3–1–1 Asahi, Matsumoto 390–8621, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Maruyama
authors.forename: Tamihide
authors.initials: T
authors.lastname: Matsunaga
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamaori
authors.forename: Sakae
authors.initials: S
authors.lastname: Sakamoto
authors.forename: Noboru
authors.initials: N
authors.lastname: Kamada
authors.forename: Katsunori
authors.initials: K
authors.lastname: Nakamura
authors.forename: Shinji
authors.initials: S
authors.lastname: Kikuchi
authors.forename: Shigeru
authors.initials: S
authors.lastname: Ohmori
mesh_terms: D000418:Albumins / Q000235:genetics; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000166:cytology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D000882:Haplorhini; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D000077297:Pregnane X Receptor; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics; D011987:Receptors, Steroid / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology; D013250:Steroid Hydroxylases / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000235:genetics
chemical_list: D000418:Albumins; D000077297:Pregnane X Receptor; D018336:Receptors, Aryl Hydrocarbon; D011987:Receptors, Steroid; D000509:alpha-Fetoproteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C022657:steroid hormone 6-beta-hydroxylase; D012293:Rifampin