Hepatocyte differentiation from human ES cells using the simple embryoid body formation method and the staged-additional cocktail.
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.
Sasaki
Katsunori
K
Department of Histology and Embryology, Shinshu University School of Medicine, Nagano 390-8621, Japan. katsmd@shinshu-u.ac.jp
Ichikawa
Hinako
H
Takei
Shunsuke
S
No
Hee Sung
HS
Tomotsune
Daihachiro
D
Kano
Yoshiya
Y
Yokoyama
Tadayuki
T
Sirasawa
Sakiko
S
Mogi
Akimi
A
Yoshie
Susumu
S
Sasaki
Shujiro
S
Yamada
Satoshi
S
Matsumoto
Ken
K
Mizuguchi
Masahiro
M
Yue
Fengming
F
Tanaka
Yoshiki
Y
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
D017931:DNA Primers