Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

Alejandro Soto-Gutiérrez, Nalu Navarro-Alvarez, Debiao Zhao, Jorge D Rivas-Carrillo, Jane Lebkowski, Noriaki Tanaka, Ira J Fox, Naoya Kobayashi

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)347-56
Number of pages10
JournalNature Protocols
Volume2
Issue number2
DOIs
Publication statusPublished - 2007

Keywords

  • Activins
  • Animals
  • Cell Culture Techniques
  • Cell Differentiation
  • Embryonic Induction
  • Embryonic Stem Cells
  • Fibroblast Growth Factor 2
  • Hepatocytes
  • Humans
  • Intercellular Signaling Peptides and Proteins
  • Mice

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