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.
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Acknowledgements
This research was supported in part by a Grant-in-Aid for Scientific Research (B) of the Japan Society for the Promotion of Science to N.K. and NIH grant DK48794 to I.J.F.
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Supplementary information
Supplementary Fig. 1
Map of pALB-GFP (11231 bps, Amp+) (PDF 5910 kb)
Supplementary Fig. 2
The sequence of pALB-GFP (11231 bp) (PDF 63 kb)
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Soto-Gutiérrez, A., Navarro-Álvarez, N., Zhao, D. et al. Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines. Nat Protoc 2, 347–356 (2007). https://doi.org/10.1038/nprot.2007.18
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DOI: https://doi.org/10.1038/nprot.2007.18
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