title: Feeder-free and serum-free production of hepatocytes, cholangiocytes, and their proliferating progenitors from human pluripotent stem cells: application to liver-specific functional and cytotoxic assays.
abstract: We have established a serum- and feeder-free culture system for the efficient differentiation of multifunctional hepatocytes from human embryonic stem (ES) cells and three entirely different induced pluripotent stem (iPS) cells (including vector/transgene-free iPS cells generated using Sendai virus vector) without cell sorting and gene manipulation. The differentiation-inducing protocol consisted of a first stage; endoderm induction, second stage; hepatic initiation, and third stage; hepatic maturation. At the end of differentiation culture, hepatocytes induced from human pluripotent stem cells expressed hepatocyte-specific proteins, such as α-fetoprotein, albumin, α1 antitrypsin and cytochrome P450 (CYP3A4), at similar or higher levels compared with three control human hepatocyte or hepatic cell lines. These human iPS/ES cell-derived hepatocytes also showed mature hepatocyte functions: indocyanine green dye uptake (≈ 30%), storage of glycogen (>80%) and metabolic activity of CYP3A4. Furthermore, they produced a highly sensitive hepatotoxicity assay system for D-galactosamine as determined by the extracellular release of hepatocyte-specific enzymes. Hepatoprotective prostaglandin E1 attenuated this toxicity. Interestingly, bile duct-specific enzymes were also detected after drug treatment, suggesting the presence of bile-duct epithelial cells (cholangiocytes) in our culture system. Electron microscopic studies confirmed the existence of cholangiocytes, and an immunostaining study proved the presence of bipotential hepatoblasts with high potential for proliferation. Differentiated cells were transferrable onto new dishes, on which small-sized proliferating cells with hepatocyte markers emerged and expanded. Thus, our differentiation culture system provides mature functional hepatocytes, cholangiocytes, and their progenitors with proliferative potential from a wide variety of human pluripotent stem cells.
authors.affiliation: Department of Disease Control, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Nakamura
authors.forename: Kumiko
authors.initials: K
authors.lastname: Saeki
authors.forename: Masami
authors.initials: M
authors.lastname: Mitsumoto
authors.forename: Satoko
authors.initials: S
authors.lastname: Matsuyama
authors.forename: Miwako
authors.initials: M
authors.lastname: Nishio
authors.forename: Koichi
authors.initials: K
authors.lastname: Saeki
authors.forename: Mamoru
authors.initials: M
authors.lastname: Hasegawa
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Miyagawa
authors.forename: Hajime
authors.initials: H
authors.lastname: Ohkita
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Kiyokawa
authors.forename: Masashi
authors.initials: M
authors.lastname: Toyoda
authors.forename: Hidenori
authors.initials: H
authors.lastname: Akutsu
authors.forename: Akihiro
authors.initials: A
authors.lastname: Umezawa
authors.forename: Akira
authors.initials: A
authors.lastname: Yuo
mesh_terms: D001652:Bile Ducts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods* / Q000706:statistics & numerical data; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D003603:Cytotoxins / Q000494:pharmacology; D061252:Feeder Cells / Q000166:cytology / Q000502:physiology; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008099:Liver / Q000166:cytology / Q000502:physiology; D008111:Liver Function Tests / Q000379:methods; D009928:Organ Specificity; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*
chemical_list: D016895:Culture Media, Serum-Free; D003603:Cytotoxins