Article | Published:

Efficient differentiation of human embryonic stem cells to definitive endoderm

Nature Biotechnology volume 23, pages 15341541 (2005) | Download Citation

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & From fertilization to gastrulation: axis formation in the mouse embryo. Curr. Opin. Genet. Dev. 11, 384–392 (2001).

  2. 2.

    & Gastrula organiser and embryonic patterning in the mouse. Semin. Cell Dev. Biol. 15, 543–554 (2004).

  3. 3.

    & Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech. Dev. 120, 1351–1383 (2003).

  4. 4.

    et al. Lack of beta-catenin affects mouse development at gastrulation. Development 121, 3529–3537 (1995).

  5. 5.

    et al. Requirement for Wnt3 in vertebrate axis formation. Nat. Genet. 22, 361–365 (1999).

  6. 6.

    , & The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice. Development 131, 2803–2815 (2004).

  7. 7.

    et al. A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. Development 120, 1919–1928 (1994).

  8. 8.

    et al. Nodal signalling in the epiblast patterns the early mouse embryo. Nature 411, 965–969 (2001).

  9. 9.

    , & Genetic dissection of nodal function in patterning the mouse embryo. Development 128, 1831–1843 (2001).

  10. 10.

    , , , & Cell fate decisions within the mouse organizer are governed by graded Nodal signals. Genes Dev. 17, 1646–1662 (2003).

  11. 11.

    The transforming growth factor-beta superfamily of receptors. Cytokine Growth Factor Rev. 15, 1–11 (2004).

  12. 12.

    et al. Development of definitive endoderm from embryonic stem cells in culture. Development 131, 1651–1662 (2004).

  13. 13.

    & Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 118, 47–59 (1993).

  14. 14.

    et al. The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins. Development 119, 1301–1315 (1993).

  15. 15.

    , , & Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 alpha, beta and gamma genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm. Development 119, 567–578 (1993).

  16. 16.

    , , , & Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4. Dev. Biol. 213, 442–456 (1999).

  17. 17.

    A glimpse into the molecular entrails of endoderm formation. Genes Dev. 16, 893–907 (2002).

  18. 18.

    et al. Depletion of definitive gut endoderm in Sox17-null mutant mice. Development 129, 2367–2379 (2002).

  19. 19.

    et al. Gastrulation in the mouse: the role of the homeobox gene goosecoid. Cell 69, 1097–1106 (1992).

  20. 20.

    et al. Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo. Development 129, 3597–3608 (2002).

  21. 21.

    & Mml, a mouse Mix-like gene expressed in the primitive streak. Mech. Dev. 87, 189–192 (1999).

  22. 22.

    , & Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343, 657–659 (1990).

  23. 23.

    et al. Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during early mesodermal patterning in mouse embryos. Development 116, 1123–1136 (1992).

  24. 24.

    & Differential localization of Mox-1 and Mox-2 proteins indicates distinct roles during development. Int. J. Dev. Biol. 40, 1179–1184 (1996).

  25. 25.

    , , & A role for SOX1 in neural determination. Development 125, 1967–1978 (1998).

  26. 26.

    et al. The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation. Dev. Biol. 182, 299–313 (1997).

  27. 27.

    et al. Overlapping and distinct expression domains of Zic2 and Zic3 during mouse gastrulation. Gene Expr. Patterns 4, 505–511 (2004).

  28. 28.

    et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264 (2002).

  29. 29.

    et al. Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J. Cell Sci. 117, 1269–1280 (2004).

  30. 30.

    , , & fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev. 8, 3032–3044 (1994).

  31. 31.

    et al. Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Dev. 8, 3045–3057 (1994).

  32. 32.

    , , & Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 13, 1834–1846 (1999).

  33. 33.

    , , , & Embryonic expression of Lim-1, the mouse homolog of Xenopus Xlim-1, suggests a role in lateral mesoderm differentiation and neurogenesis. Dev. Biol. 161, 168–178 (1994).

  34. 34.

    & Requirement for Lim1 in head-organizer function. Nature 374, 425–430 (1995).

  35. 35.

    & Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114, 755–768 (1992).

  36. 36.

    , , & Expression and function of FGF-4 in peri-implantation development in mouse embryos. Development 120, 2259–2269 (1994).

  37. 37.

    et al. Cerberus-like is a secreted factor with neutralizing activity expressed in the anterior primitive endoderm of the mouse gastrula. Mech. Dev. 68, 45–57 (1997).

  38. 38.

    et al. Murine cerberus homologue mCer-1: a candidate anterior patterning molecule. Dev. Biol. 194, 135–151 (1998).

  39. 39.

    , & Expression of the mouse cerberus-related gene, Cerr1, suggests a role in anterior neural induction and somitogenesis. Proc. Natl. Acad. Sci. USA 95, 6198–6203 (1998).

  40. 40.

    , & A mouse cerberus/Dan-related gene family. Dev. Biol. 209, 98–110 (1999).

  41. 41.

    , , & The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate mesoderm. Development 128, 155–166 (2001).

  42. 42.

    , & Differences in the embryonic expression patterns of mouse Foxf1 and -2 match their distinct mutant phenotypes. Dev. Dyn. 229, 328–333 (2004).

  43. 43.

    et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376, 62–66 (1995).

  44. 44.

    , , & Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 2105–2116 (1995).

  45. 45.

    et al. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 13, 424–436 (1999).

  46. 46.

    & Mouse Cdx-1 expression during gastrulation. Development 117, 191–203 (1993).

  47. 47.

    & Expression of homeobox genes, including an insulin promoting factor, in the murine yolk sac at the time of hematopoietic initiation. Mol. Reprod. Dev. 48, 145–153 (1997).

  48. 48.

    et al. Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst. Development 132, 2093–2102 (2005).

  49. 49.

    , , , & Placental growth hormones. Endocrine 19, 73–79 (2002).

  50. 50.

    et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat. Cell Biol. 2, 84–89 (2000).

  51. 51.

    & Expression of N-cadherin adhesion molecules associated with early morphogenetic events in chick development. Nature 320, 447–449 (1986).

  52. 52.

    et al. Developmental defects in mouse embryos lacking N-cadherin. Dev. Biol. 181, 64–78 (1997).

  53. 53.

    & Tissue specificity of alpha-fetoprotein messenger RNA expression during mouse embryogenesis. EMBO J. 2, 549–554 (1983).

  54. 54.

    , , , & Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos. Development 122, 2271–2281 (1996).

  55. 55.

    , , , & Chimeric analysis of fibroblast growth factor receptor-1 (Fgfr1) function: a role for FGFR1 in morphogenetic movement through the primitive streak. Development 124, 2829–2841 (1997).

  56. 56.

    et al. Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 132, 4363–4374 (2005).

  57. 57.

    et al. Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. Stem Cells 23, 166–185 (2005).

  58. 58.

    , & Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403–421 (2004).

  59. 59.

    , , & Feeder layer- and serum-free culture of human embryonic stem cells. Biol. Reprod. 70, 837–845 (2004).

  60. 60.

    et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23, 489–495 (2005).

  61. 61.

    , , & TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132, 1273–1282 (2005).

Download references

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.

Author information

Affiliations

  1. CyThera Inc., 3550 General Atomics Ct., San Diego, California 92121, USA.

    • Kevin A D'Amour
    • , Alan D Agulnick
    • , Susan Eliazer
    • , Olivia G Kelly
    • , Evert Kroon
    •  & Emmanuel E Baetge

Authors

  1. Search for Kevin A D'Amour in:

  2. Search for Alan D Agulnick in:

  3. Search for Susan Eliazer in:

  4. Search for Olivia G Kelly in:

  5. Search for Evert Kroon in:

  6. Search for Emmanuel E Baetge in:

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Emmanuel E Baetge.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Validation of SOX17 anti-serum

  2. 2.

    Supplementary Fig. 2

    Exogenous activin exposure determines the proportion of mesoderm and DE

  3. 3.

    Supplementary Fig. 3

    Phase contrast images of activin and BMP4/SU5402 treated cultures

  4. 4.

    Supplementary Fig. 4

    Immunocytochemical analyses of E-cadherin, N-cadherin and brachyury during hESC differentiation

  5. 5.

    Supplementary Fig. 5

    Quantification of CXCR4-positive cell numbers by flow cytometry

  6. 6.

    Supplementary Fig. 6

    CXCR4-based isolation of hESC-DE can be applied to multiple hESC lines

  7. 7.

    Supplementary Fig. 7

    Eight hESC lines exhibit PS-like gene expression dynamics during DE formation

  8. 8.

    Supplementary Fig. 8

    Model summarizing transitions during hESC differentiation

  9. 9.

    Supplementary Table 1

    Gene expression of markers during gastrulation

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt1163

Further reading