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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.

References

  1. Lu, C.C., Brennan, J. & Robertson, E.J. From fertilization to gastrulation: axis formation in the mouse embryo. Curr. Opin. Genet. Dev. 11, 384–392 (2001).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. Kelly, O.G., Pinson, K.I. & Skarnes, W.C. The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice. Development 131, 2803–2815 (2004).

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. Lowe, L.A., Yamada, S. & Kuehn, M.R. Genetic dissection of nodal function in patterning the mouse embryo. Development 128, 1831–1843 (2001).

    CAS  PubMed  Google Scholar 

  10. Vincent, S.D., Dunn, N.R., Hayashi, S., Norris, D.P. & Robertson, E.J. Cell fate decisions within the mouse organizer are governed by graded Nodal signals. Genes Dev. 17, 1646–1662 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  15. Monaghan, A.P., Kaestner, K.H., Grau, E. & Schutz, G. 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).

    CAS  PubMed  Google Scholar 

  16. McGrath, K.E., Koniski, A.D., Maltby, K.M., McGann, J.K. & Palis, J. Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4. Dev. Biol. 213, 442–456 (1999).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  22. Wilkinson, D.G., Bhatt, S. & Herrmann, B.G. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343, 657–659 (1990).

    Article  CAS  PubMed  Google Scholar 

  23. Candia, A.F. 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).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  25. Pevny, L.H., Sockanathan, S., Placzek, M. & Lovell-Badge, R. A role for SOX1 in neural determination. Development 125, 1967–1978 (1998).

    CAS  PubMed  Google Scholar 

  26. Nagai, T. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  30. Yamaguchi, T.P., Harpal, K., Henkemeyer, M. & Rossant, J. fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev. 8, 3032–3044 (1994).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  32. Sun, X., Meyers, E.N., Lewandoski, M. & Martin, G.R. Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 13, 1834–1846 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Barnes, J.D., Crosby, J.L., Jones, C.M., Wright, C.V. & Hogan, B.L. 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).

    Article  PubMed  Google Scholar 

  34. Shawlot, W. & Behringer, R.R. Requirement for Lim1 in head-organizer function. Nature 374, 425–430 (1995).

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  36. Rappolee, D.A., Basilico, C., Patel, Y. & Werb, Z. Expression and function of FGF-4 in peri-implantation development in mouse embryos. Development 120, 2259–2269 (1994).

    CAS  PubMed  Google Scholar 

  37. Belo, J.A. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  39. Shawlot, W., Deng, J.M. & Behringer, R.R. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Pearce, J.J., Penny, G. & Rossant, J. A mouse cerberus/Dan-related gene family. Dev. Biol. 209, 98–110 (1999).

    Article  CAS  PubMed  Google Scholar 

  41. Mahlapuu, M., Ormestad, M., Enerback, S. & Carlsson, P. The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate mesoderm. Development 128, 155–166 (2001).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  44. Winnier, G., Blessing, M., Labosky, P.A. & Hogan, B.L. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev. 9, 2105–2116 (1995).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Meyer, B.I. & Gruss, P. Mouse Cdx-1 expression during gastrulation. Development 117, 191–203 (1993).

    CAS  PubMed  Google Scholar 

  47. McGrath, K.E. & Palis, J. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  49. Lacroix, M.C., Guibourdenche, J., Frendo, J.L., Pidoux, G. & Evain-Brion, D. Placental growth hormones. Endocrine 19, 73–79 (2002).

    Article  CAS  PubMed  Google Scholar 

  50. Batlle, E. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  53. Dziadek, M.A. & Andrews, G.K. Tissue specificity of alpha-fetoprotein messenger RNA expression during mouse embryogenesis. EMBO J. 2, 549–554 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Weiler-Guettler, H., Aird, W.C., Rayburn, H., Husain, M. & Rosenberg, R.D. Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos. Development 122, 2271–2281 (1996).

    CAS  PubMed  Google Scholar 

  55. Ciruna, B.G., Schwartz, L., Harpal, K., Yamaguchi, T.P. & Rossant, J. 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).

    CAS  PubMed  Google Scholar 

  56. Tada, S. 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).

    Article  CAS  PubMed  Google Scholar 

  57. Wei, C.L. 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).

    Article  CAS  PubMed  Google Scholar 

  58. Vallier, L., Reynolds, D. & Pedersen, R.A. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403–421 (2004).

    Article  CAS  PubMed  Google Scholar 

  59. Amit, M., Shariki, C., Margulets, V. & Itskovitz-Eldor, J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol. Reprod. 70, 837–845 (2004).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  61. James, D., Levine, A.J., Besser, D. & Hemmati-Brivanlou, A. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132, 1273–1282 (2005).

    Article  CAS  PubMed  Google Scholar 

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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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