题名：Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines
作者：Alejandro Soto-Guti??rrez, Nalu Navarro-??lvarez, Debiao Zhao, Jorge D Rivas-Carrillo, Jane Lebkowski, Noriaki Tanaka, Ira J Fox, Naoya Kobayashi
来源：Nature Protocols[IF=----], 2, 347 - 356 (01 Mar 2007)
摘要： Alejandro Soto-Gutirrez1,5, Nalu Navarro-lvarez1,5, Debiao Zhao2, Jorge D Rivas-Carrillo1, Jane Lebkowski3, Noriaki Tanaka1, Ira J Fox4 and Naoya Kobayashi1
1Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
2Roslin Institute, Roslin, Midlothian, EH25 9PS, U.K.
3Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025, USA.
4Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska 68198-3285, USA.
5These authors contributed equally to this work.
Correspondence to: Ira J Fox4 e-mail: email@example.com
Correspondence to: Naoya Kobayashi1 e-mail: firstname.lastname@example.org
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. 40.
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