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BMP-4 is required for hepatic specification of mouse embryonic stem cell–derived definitive endoderm

Nature Biotechnology volume 24, pages 1402–1411 (2006)Cite this article

Abstract

When differentiated in the presence of activin A in serum-free conditions, mouse embryonic stem cells efficiently generate an endoderm progenitor population defined by the coexpression of either Brachyury, Foxa2 and c-Kit, or c-Kit and Cxcr4. Specification of these progenitors with bone morphogenetic protein-4 in combination with basic fibroblast growth factor and activin A results in the development of hepatic populations highly enriched (45–70%) for cells that express the α-fetoprotein and albumin proteins. These cells also express transcripts of Afp, Alb1, Tat, Cps1, Cyp7a1 and Cyp3a11; they secrete albumin, store glycogen, show ultrastructural characteristics of mature hepatocytes, and are able to integrate into and proliferate in injured livers in vivo and mature into hepatocytes expressing dipeptidyl peptidase IV or fumarylacetoacetate hydrolase. Together, these findings establish a developmental pathway in embryonic stem cell differentiation cultures that leads to efficient generation of cells with an immature hepatocytic phenotype.

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Figure 1: Induction of GFP-Bry+/CD4-Foxa2high/c-Kithigh definitive endoderm progenitors by activin A.
Figure 2: Hepatic specification of the day 4 GFP-Bry+/CD4-Foxa2high/c-Kithigh population.
Figure 3: Effect of BMP-4 concentration on liver specification of the day 4 GFP-Bry+/CD4-Foxa2high/c-Kithigh population.
Figure 4: Immunostaining analysis of adherent cells and suspended aggregates generated from day 6 plated aggregates.
Figure 5: Functional analyses of ES cell–derived hepatic cells.
Figure 6: Generation of hepatic cells from an unmanipulated ES cell line.

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Acknowledgements

We wish to thank Ronald Gordon for expert assistance in the EM analysis and John Fallon for his expertise in pathology. We would like to thank Xiao Zhao for her helpful technical support with the real-time PCR, as well as the members of the Keller laboratory for critical reading of the manuscript. We also thank R.M. Tanguay for providing anti-mouse Fah antibody. This work was supported by US National Institutes of Health grants K01DK068041-01 (V.G.-E.), U01 DK072513 (G.K.), F32 HL076058 (P.G.), R01-DK17609 (D.A.S.) and P30-DK41296 (D.A.S.), and by the German Academic Exchange Service DAAD (D.N.) and the Deutsche Forschungsgemeinschaft K03478/1-1 (C.I.K.).

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  1. Atsushi Kubo

    Present address: First Department of Internal Medicine, Nara Medical University, Nara, 634-8521, Japan

Authors and Affiliations

  1. Department of Gene and Cell Medicine, Black Family Stem Cell Institute, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, 10029, New York, USA

    Valerie Gouon-Evans, Lise Boussemart, Paul Gadue & Gordon Keller

  2. Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, New York, 10461, New York, USA

    Dirk Nierhoff, Christoph I Koehler & David A Shafritz

  3. Department of Gastroenterology and Hepatology, University of Cologne, Germany

    Dirk Nierhoff

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Contributions

V.G.-E. and L.B. contributed to ES cell differentiation into hepatic cells and their characterization in vitro, P.G. conributed to making the hCD4 knock-in into the Foxa2 locus in the GFP-Bry ES cell line, D.N. and C.I.K. transplanted the ES cell-derived hepatic cells into both mouse models and analyzed their livers. C.I.K. provided the hepatoblast and hepatocyte control cell samples for in vitro assays, A.K. initiated the characterization of the GFP-Bry+/c-Kithigh population as definitive endoderm progenitors following activin A induction, D.A.S. contributed to the planning and design of the in vivo studies, G.K. and V.G.-E. contributed to the planning and design of the project and manuscript writing.

Corresponding author

Correspondence to Gordon Keller.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Quantification of afp+ and albumin+ cell development (PDF 132 kb)

Supplementary Fig. 2

Summary of the two basic protocols used for the generation of hepatic cells from ES cells. (PDF 1033 kb)

Supplementary Table 1

Proliferative potential of endoderm progenitor populations specified with different combinations of factors. (PDF 24 kb)

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Gouon-Evans, V., Boussemart, L., Gadue, P. et al. BMP-4 is required for hepatic specification of mouse embryonic stem cell–derived definitive endoderm. Nat Biotechnol 24, 1402–1411 (2006). https://doi.org/10.1038/nbt1258

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