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Efficient differentiation of human embryonic stem cells to definitive endoderm

Abstract

The potential of human embryonic stem (hES) cells to differentiate into cell types of a variety of organs has generated much excitement over the possible use of hES cells in therapeutic applications. Of great interest are organs derived from definitive endoderm, such as the pancreas. We have focused on directing hES cells to the definitive endoderm lineage as this step is a prerequisite for efficient differentiation to mature endoderm derivatives. Differentiation of hES cells in the presence of activin A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent of vertebrate gastrulation. These findings may facilitate the use of hES cells for therapeutic purposes and as in vitro models of development.

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Figure 1: Effect of serum concentration on hES cell–derived definitive endoderm production.
Figure 2: The temporal dynamics of gene expression indicates transition through a primitive streak–like intermediate before expression of definitive endoderm genes in activin A but not BMP4/SU5402-treated cultures.
Figure 3: Kinetics of mRNA and protein expression determined by real-time Q-PCR, western blotting and immunocytochemistry.
Figure 4: Immunocytochemical analyses of differentiating hES cells are consistent with an EMT.
Figure 5: Isolation of CXCR4-positive cells using FACS further enriches hES cell–derived definitive endoderm to near homogeneity.
Figure 6: Endodermal differentiation of hES cell–derived definitive endoderm grafts.

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Acknowledgements

We wish to acknowledge Melissa Carpenter for culture and differentiation of H7 and H9 hES cells, Gillian Beattie for culture and differentiation of HUES7 hES cells and Bobbie Daughters and Jie Zheng for kidney capsule implantation and histology, respectively. Special thanks to Melissa Carpenter, Anne Bang, Matthias Hebrok, Didier Stainier and Jim Wells for helpful advice and critical review of the manuscript.

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Correspondence to Emmanuel E Baetge.

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Supplementary information

Supplementary Fig. 1

Validation of SOX17 anti-serum (PDF 1136 kb)

Supplementary Fig. 2

Exogenous activin exposure determines the proportion of mesoderm and DE (PDF 630 kb)

Supplementary Fig. 3

Phase contrast images of activin and BMP4/SU5402 treated cultures (PDF 2264 kb)

Supplementary Fig. 4

Immunocytochemical analyses of E-cadherin, N-cadherin and brachyury during hESC differentiation (PDF 732 kb)

Supplementary Fig. 5

Quantification of CXCR4-positive cell numbers by flow cytometry (PDF 845 kb)

Supplementary Fig. 6

CXCR4-based isolation of hESC-DE can be applied to multiple hESC lines (PDF 650 kb)

Supplementary Fig. 7

Eight hESC lines exhibit PS-like gene expression dynamics during DE formation (PDF 839 kb)

Supplementary Fig. 8

Model summarizing transitions during hESC differentiation (PDF 454 kb)

Supplementary Table 1

Gene expression of markers during gastrulation (PDF 20 kb)

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D'Amour, K., Agulnick, A., Eliazer, S. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23, 1534–1541 (2005). https://doi.org/10.1038/nbt1163

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