title: Hepatocyte differentiation from human ES cells using the simple embryoid body formation method and the staged-additional cocktail.
abstract: To induce hepatocytes from human embryonic stem (hES) cells easily and effectively, a simple suspension culture method that separates ES colonies with a scraper and transfers them into newly developed, nonadherent MPC (2-methacryloyloxyethyl phosphorylcholine) plates, and the staged-additional cocktail method, including growth factors, cytokines, and Lanford serum-free medium, were developed and evaluated mainly by morphological analysis. The formed embryoid bodies (EBs) showed compact cellular agglomeration until day 4 and later formed coeloms in their interior. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis showed that they are gene markers of the three germ layers. Mesenchymal cells with rough endoplasmic reticulum (rER) and extracellular matrix (ECM), and without junctions, were recognized in the interior of the EBs by transmission electron microscopy (TEM) in addition to epithelial cells. When they were stimulated by the staged-additional cocktail, they expressed albumin-positive immunoreactivity, indocyanine green (ICG) uptake, and typical ultrastructures of the hepatocytes, including bile canaliculi. These results indicate that these combined methods promote EB formation and hepatocyte differentiation from hES cells.
authors.affiliation: Department of Histology and Embryology, Shinshu University School of Medicine, Nagano 390-8621, Japan. katsmd@shinshu-u.ac.jp
authors.forename: Katsunori
authors.initials: K
authors.lastname: Sasaki
authors.forename: Hinako
authors.initials: H
authors.lastname: Ichikawa
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Takei
authors.forename: Hee Sung
authors.initials: HS
authors.lastname: No
authors.forename: Daihachiro
authors.initials: D
authors.lastname: Tomotsune
authors.forename: Yoshiya
authors.initials: Y
authors.lastname: Kano
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Yokoyama
authors.forename: Sakiko
authors.initials: S
authors.lastname: Sirasawa
authors.forename: Akimi
authors.initials: A
authors.lastname: Mogi
authors.forename: Susumu
authors.initials: S
authors.lastname: Yoshie
authors.forename: Shujiro
authors.initials: S
authors.lastname: Sasaki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamada
authors.forename: Ken
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Masahiro
authors.initials: M
authors.lastname: Mizuguchi
authors.forename: Fengming
authors.initials: F
authors.lastname: Yue
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Tanaka
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000648:ultrastructure; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers