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Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell–derived hepatocytes

A Corrigendum to this article was published on 01 February 2007

Abstract

Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter–based cell sorting. ES cell–derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell–derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell–derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.

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Figure 1: Schematic representation of the strategy for differentiation of mouse embryonic stem cells to hepatocytes and their use for BAL therapy.
Figure 2: Characterization and purification of mouse ES cell-derived hepatocytes.
Figure 3: Immunohistochemical examination of ALB and AFP expression in differentiating mES cells and morphological and functional analyses of ES-Heps in vitro.
Figure 4: Fate of ES-Heps in long-term culture.
Figure 5: Ammonia, hepatic encephalopathy, blood glucose, and survival of hepatectomized mice after BAL therapy.
Figure 6: Morphologic and histological analysis of ES-Heps in BAL modules.

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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 National Institutes of Health grant DK48794 to I.J.F. We thank Donna B. Stolz for useful comments for the paper. We thank Ann Kyle for editorial assistance.

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Authors and Affiliations

Authors

Contributions

Ira J. Fox and Naoya Kobayashi had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analyses. Study concept and design, I.J.F., N.K., A.S.-G., J.L., J.-W.Y., H.-S.J., N.T. Acquisition of data, I.J.F., N.K., A.S.-G., N.T. Analysis and interpretation of data, I.J.F., N.K., A.S.-G. Drafting of the manuscript, I.J.F., N.K., A.S.-G., J.L., J.-W.Y., H.-S.J. Critical revision of the manuscript for important intellectual content, I.J.F., N.K., A.S.-G. Statistical analysis, T.O., H.N. Obtained funding, I.J.F., N.K. Administrative, technical, or material support, I.J.F., N.K., A.S.-G., N.N.-A., J.D.R.-C., H.B., T.U., Y.T., D.Z., Y.C., K.T., M.N., A.M. Study supervision, I.J.F., N.K. All authors contributed to the preparation of the report.

Corresponding authors

Correspondence to Naoya Kobayashi or Ira J Fox.

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Competing interests

Jane Lebkowski is an employee of Geron Corporation.

Supplementary information

Supplementary Fig. 1

Characteristics of differentiating ES-Heps by RT-PCR. (PDF 180 kb)

Supplementary Fig. 2

Differentiation and functional assessment of ES-Heps cultured with different combinations of liver nonparenchymal cell lines. (PDF 76 kb)

Supplementary Fig. 3

Expression of pancreatic exocrine factors and hormones by ES-Heps. (PDF 244 kb)

Supplementary Fig. 4

Characteristics of the implantable bio-artificial live (BAL) module. (PDF 117 kb)

Supplementary Fig. 5

Ammonia blood levels, glucose blood levels, and survival of the 90% hepatectomized mice after BAL therapy using different ES-Hep cell numbers. (PDF 102 kb)

Supplementary Fig. 6

Tissue blood flow at the site of the implanted BAL module. (PDF 168 kb)

Supplementary Fig. 7

Expression of primitive endoderm and hepatocyte-specific genes in 2-day-old embryonic bodies (D0 cells) using immunohistochemistry. (PDF 657 kb)

Supplementary Fig. 8

Gene expression profile during liver-specific ES cell differentiation: effect of culture in activin A and bFGF. (PDF 165 kb)

Supplementary Fig. 9

Cytokines and growth factors produced by the human liver nonparenchymal cell lines. (PDF 409 kb)

Supplementary Fig. 10

Efficacy of dHGF versus full-length HGF in hepatic differentiation of mouse ES cells. (PDF 81 kb)

Supplementary Fig. 11

Hepatic differentiation of mouse ES cells using different culture substrates. (PDF 168 kb)

Supplementary Fig. 12

Growth of differentiating mouse ES cells toward ES-Heps under serum-free conditions. (PDF 100 kb)

Supplementary Table 1

Differential expression of albumin (ALB) and alpha-feto protein (AFP) in differentiating mES cells. (PDF 26 kb)

Supplementary Table 2

Full names of cytokines and growth factors produced by human liver nonparenchymal cell lines as assessed by protein array. (PDF 82 kb)

Supplementary Methods (PDF 35 kb)

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Soto-Gutiérrez, A., Kobayashi, N., Rivas-Carrillo, J. et al. Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell–derived hepatocytes. Nat Biotechnol 24, 1412–1419 (2006). https://doi.org/10.1038/nbt1257

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