Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines.
This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.
Soto-Gutiérrez
Alejandro
A
Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Navarro-Alvarez
Nalu
N
Zhao
Debiao
D
Rivas-Carrillo
Jorge D
JD
Lebkowski
Jane
J
Tanaka
Noriaki
N
Fox
Ira J
IJ
Kobayashi
Naoya
N
D028341:Activins; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D004627:Embryonic Induction / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology; D016222:Fibroblast Growth Factor 2; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice
D036341:Intercellular Signaling Peptides and Proteins; C422405:activin A; D016222:Fibroblast Growth Factor 2; D028341:Activins