Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Production of pancreatic hormone–expressing endocrine cells from human embryonic stem cells

Nature Biotechnology volume 24, pages 1392–1401 (2006)Cite this article

Abstract

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor—en route to cells that express endocrine hormones. The hES cell–derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal β-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell–derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic of differentiation procedure and protein expression for some key markers of pancreatic differentiation.
Figure 2: Immunofluorescence analysis of transitions from definitive endoderm to gut tube and from gut tube to posterior foregut.
Figure 3: Immunofluorescence analysis of transitions from pancreas to endocrine precursor and from endocrine precursor to hormone-expressing cells.
Figure 4: Immunofluorescence analysis of hormone-expressing cells.
Figure 5: Flow cytometry analysis of insulin-expressing cells.
Figure 6: High insulin content and robust C-peptide release.

Similar content being viewed by others

References

  1. Hoffman, L.M. & Carpenter, M.K. Characterization and culture of human embryonic stem cells. Nat. Biotechnol. 23, 699–708 (2005).

    Article  CAS  Google Scholar 

  2. Liew, C.G. et al. Human embryonic stem cells: possibilities for human cell transplantation. Ann. Med. 37, 521–532 (2005).

    Article  CAS  Google Scholar 

  3. Bonner-Weir, S. & Weir, G.C. New sources of pancreatic beta-cells. Nat. Biotechnol. 23, 857–861 (2005).

    Article  CAS  Google Scholar 

  4. Madsen, O.D. Stem cells and diabetes treatment. APMIS 113, 858–875 (2005).

    Article  Google Scholar 

  5. Assady, S. et al. Insulin production by human embryonic stem cells. Diabetes 50, 1691–1697 (2001).

    Article  CAS  Google Scholar 

  6. Segev, H., Fishman, B., Ziskind, A., Shulman, M. & Itskovitz-Eldor, J. Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells 22, 265–274 (2004).

    Article  CAS  Google Scholar 

  7. Baharvand, H., Jafary, H., Massumi, M. & Ashtiani, S.K. Generation of insulin-secreting cells from human embryonic stem cells. Dev. Growth Differ. 48, 323–332 (2006).

    Article  CAS  Google Scholar 

  8. Xu, X. et al. Endoderm and pancreatic islet lineage differentiation from human embryonic stem cells. Cloning Stem Cells 8, 96–107 (2006).

    Article  CAS  Google Scholar 

  9. Kwon, Y.D. et al. Cellular manipulation of human embryonic stem cells by TAT-PDX1 protein transduction. Mol. Ther. 12, 28–32 (2005).

    Article  CAS  Google Scholar 

  10. Hori, Y., Gu, X., Xie, X. & Kim, S.K. Differentiation of insulin-producing cells from human neural progenitor cells. PLoS Med. 2, e103 (2005).

    Article  Google Scholar 

  11. Roche, E., Sepulcre, P., Reig, J.A., Santana, A. & Soria, B. Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells. FASEB J. 19, 1341–1343 (2005).

    Article  CAS  Google Scholar 

  12. Sipione, S., Eshpeter, A., Lyon, J.G., Korbutt, G.S. & Bleackley, R.C. Insulin expressing cells from differentiated embryonic stem cells are not beta cells. Diabetologia 47, 499–508 (2004).

    Article  CAS  Google Scholar 

  13. Rajagopal, J., Anderson, W.J., Kume, S., Martinez, O.I. & Melton, D.A. Insulin staining of ES cell progeny from insulin uptake. Science 299, 363 (2003).

    Google Scholar 

  14. Hansson, M. et al. Artifactual insulin release from differentiated embryonic stem cells. Diabetes 53, 2603–2609 (2004).

    Article  CAS  Google Scholar 

  15. Tam, P.P., Williams, E.A. & Chan, W.Y. Gastrulation in the mouse embryo: ultrastructural and molecular aspects of germ layer morphogenesis. Microsc. Res. Tech. 26, 301–328 (1993).

    Article  CAS  Google Scholar 

  16. Wells, J.M. & Melton, D.A. Vertebrate endoderm development. Annu. Rev. Cell Dev. Biol. 15, 393–410 (1999).

    Article  CAS  Google Scholar 

  17. Stafford, D., Hornbruch, A., Mueller, P.R. & Prince, V.E. A conserved role for retinoid signaling in vertebrate pancreas development. Dev. Genes Evol. 214, 432–441 (2004).

    Article  CAS  Google Scholar 

  18. Lau, J., Kawahira, H. & Hebrok, M. Hedgehog signaling in pancreas development and disease. Cell. Mol. Life Sci. 63, 642–652 (2006).

    Article  CAS  Google Scholar 

  19. Gu, G., Dubauskaite, J. & Melton, D.A. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129, 2447–2457 (2002).

    CAS  Google Scholar 

  20. Wilson, M.E., Scheel, D. & German, M.S. Gene expression cascades in pancreatic development. Mech. Dev. 120, 65–80 (2003).

    Article  CAS  Google Scholar 

  21. Bhushan, A. et al. Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis. Development 128, 5109–5117 (2001).

    CAS  Google Scholar 

  22. Murtaugh, L.C. & Melton, D.A. Genes, signals, and lineages in pancreas development. Annu. Rev. Cell Dev. Biol. 19, 71–89 (2003).

    Article  CAS  Google Scholar 

  23. Jensen, J. Gene regulatory factors in pancreatic development. Dev. Dyn. 229, 176–200 (2004).

    Article  CAS  Google Scholar 

  24. D'Amour, K.A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534–1541 (2005).

    Article  CAS  Google Scholar 

  25. Yamaguchi, T.P. Heads or tails: Wnts and anterior-posterior patterning. Curr. Biol. 11, R713–R724 (2001).

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

  27. Yasunaga, M. et al. Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat. Biotechnol. 23, 1542–1550 (2005).

    Article  CAS  Google Scholar 

  28. 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  Google Scholar 

  29. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  31. Barbacci, E. et al. Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification. Development 126, 4795–4805 (1999).

    CAS  Google Scholar 

  32. Coffinier, C., Barra, J., Babinet, C. & Yaniv, M. Expression of the vHNF1/HNF1β homeoprotein gene during mouse organogenesis. Mech. Dev. 89, 211–213 (1999).

    Article  CAS  Google Scholar 

  33. Duncan, S.A. et al. Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo: HNF-4 is a marker for primary endoderm in the implanting blastocyst. Proc. Natl. Acad. Sci. USA 91, 7598–7602 (1994).

    Article  CAS  Google Scholar 

  34. Sun, Z. & Hopkins, N. vhnf1, the MODY5 and familial GCKD-associated gene, regulates regional specification of the zebrafish gut, pronephros, and hindbrain. Genes Dev. 15, 3217–3229 (2001).

    Article  CAS  Google Scholar 

  35. Chen, Y. et al. Retinoic acid signaling is essential for pancreas development and promotes endocrine at the expense of exocrine cell differentiation in Xenopus. Dev. Biol. 271, 144–160 (2004).

    Article  CAS  Google Scholar 

  36. Stafford, D. & Prince, V.E. Retinoic acid signaling is required for a critical early step in zebrafish pancreatic development. Curr. Biol. 12, 1215–1220 (2002).

    Article  CAS  Google Scholar 

  37. Molotkov, A., Molotkova, N. & Duester, G. Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development. Dev. Dyn. 232, 950–957 (2005).

    Article  CAS  Google Scholar 

  38. Martin, M. et al. Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice. Dev. Biol. 284, 399–411 (2005).

    Article  CAS  Google Scholar 

  39. Hebrok, M., Kim, S.K. & Melton, D.A. Notochord repression of endodermal Sonic hedgehog permits pancreas development. Genes Dev. 12, 1705–1713 (1998).

    Article  CAS  Google Scholar 

  40. Rindi, G., Leiter, A.B., Kopin, A.S., Bordi, C. & Solcia, E. The “normal” endocrine cell of the gut: changing concepts and new evidences. Ann. NY Acad. Sci. 1014, 1–12 (2004).

    Article  CAS  Google Scholar 

  41. Tomita, T. New markers for pancreatic islets and islet cell tumors. Pathol. Int. 52, 425–432 (2002).

    Article  Google Scholar 

  42. Latif, Z.A., Noel, J. & Alejandro, R. A simple method of staining fresh and cultured islets. Transplantation 45, 827–830 (1988).

    Article  CAS  Google Scholar 

  43. Frederickson, C. Imaging zinc: old and new tools. Sci. STKE 2003, pe18 (2003).

    Google Scholar 

  44. Sturgess, N.C., Ashford, M.L., Cook, D.L. & Hales, C.N. The sulphonylurea receptor may be an ATP-sensitive potassium channel. Lancet 2, 474–475 (1985).

    Article  CAS  Google Scholar 

  45. Misler, S. et al. Stimulus-secretion coupling in beta-cells of transplantable human islets of Langerhans. Evidence for a critical role for Ca2+ entry. Diabetes 41, 662–670 (1992).

    Article  CAS  Google Scholar 

  46. Pyne, N.J. & Furman, B.L. Cyclic nucleotide phosphodiesterases in pancreatic islets. Diabetologia 46, 1179–1189 (2003).

    Article  CAS  Google Scholar 

  47. Prentki, M. & Matschinsky, F.M. Ca2+, cAMP, and phospholipid-derived messengers in coupling mechanisms of insulin secretion. Physiol. Rev. 67, 1185–1248 (1987).

    Article  CAS  Google Scholar 

  48. Klimstra, D.S. Pancreas. in Histology for Pathologists (ed. Sternberg, S.S.) 613–647 (Lippincott-Raven Publishers, Philadelphia, 1997).

    Google Scholar 

  49. McLean, A.B. et al. Activin A efficiently specifies definitive endoderm from human embryonic stem cells only when PI3K signaling is suppressed. Stem Cells (in the press).

  50. Hart, A., Papadopoulou, S. & Edlund, H. Fgf10 maintains notch activation, stimulates proliferation, and blocks differentiation of pancreatic epithelial cells. Dev. Dyn. 228, 185–193 (2003).

    Article  CAS  Google Scholar 

  51. Ye, F., Duvillie, B. & Scharfmann, R. Fibroblast growth factors 7 and 10 are expressed in the human embryonic pancreatic mesenchyme and promote the proliferation of embryonic pancreatic epithelial cells. Diabetologia 48, 277–281 (2005).

    Article  CAS  Google Scholar 

  52. Jacquemin, P. et al. An endothelial-mesenchymal relay pathway regulates early phases of pancreas development. Dev. Biol. 290, 189–199 (2006).

    Article  CAS  Google Scholar 

  53. Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371, 606–609 (1994).

    Article  CAS  Google Scholar 

  54. Jacquemin, P., Lemaigre, F.P. & Rousseau, G.G. The Onecut transcription factor HNF-6 (OC-1) is required for timely specification of the pancreas and acts upstream of Pdx-1 in the specification cascade. Dev. Biol. 258, 105–116 (2003).

    Article  CAS  Google Scholar 

  55. Harrison, K.A., Thaler, J., Pfaff, S.L., Gu, H. & Kehrl, J.H. Pancreas dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9-deficient mice. Nat. Genet. 23, 71–75 (1999).

    Article  CAS  Google Scholar 

  56. Li, H., Arber, S., Jessell, T.M. & Edlund, H. Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9. Nat. Genet. 23, 67–70 (1999).

    Article  CAS  Google Scholar 

  57. Schwitzgebel, V.M. et al. Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development 127, 3533–3542 (2000).

    CAS  Google Scholar 

  58. Gradwohl, G., Dierich, A., LeMeur, M. & Guillemot, F. neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc. Natl. Acad. Sci. USA 97, 1607–1611 (2000).

    Article  CAS  Google Scholar 

  59. Teitelman, G., Alpert, S., Polak, J.M., Martinez, A. & Hanahan, D. Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon and the neuronal proteins tyrosine hydroxylase and neuropeptide Y, but not pancreatic polypeptide. Development 118, 1031–1039 (1993).

    CAS  Google Scholar 

  60. Polak, M., Bouchareb-Banaei, L., Scharfmann, R. & Czernichow, P. Early pattern of differentiation in the human pancreas. Diabetes 49, 225–232 (2000).

    Article  CAS  Google Scholar 

  61. De Krijger, R.R. et al. The midgestational human fetal pancreas contains cells coexpressing islet hormones. Dev. Biol. 153, 368–375 (1992).

    Article  CAS  Google Scholar 

  62. Herrera, P.L., Nepote, V. & Delacour, A. Pancreatic cell lineage analyses in mice. Endocrine 19, 267–278 (2002).

    Article  CAS  Google Scholar 

  63. Slack, J.M. Developmental biology of the pancreas. Development 121, 1569–1580 (1995).

    CAS  Google Scholar 

  64. Pictet, R.L. & Rutter, W.J. Development of the embryonic endocrine pancreas. in Handbook of Physiology (eds. Steiner, D.F. & Frenkel, N.) 25–66 (Williams and Wilkins, Washington, DC, 1972).

    Google Scholar 

  65. Schisler, J.C. et al. The Nkx6.1 homeodomain transcription factor suppresses glucagon expression and regulates glucose-stimulated insulin secretion in islet beta cells. Proc. Natl. Acad. Sci. USA 102, 7297–7302 (2005).

    Article  CAS  Google Scholar 

  66. Nishimura, W. et al. A switch from MafB to MafA expression accompanies differentiation to pancreatic beta-cells. Dev. Biol. 293, 526–539 (2006).

    Article  CAS  Google Scholar 

  67. Demeterco, C., Beattie, G.M., Dib, S.A., Lopez, A.D. & Hayek, A. A role for activin A and betacellulin in human fetal pancreatic cell differentiation and growth. J. Clin. Endocrinol. Metab. 85, 3892–3897 (2000).

    CAS  Google Scholar 

  68. Hayek, A. & Beattie, G.M. Experimental transplantation of human fetal and adult pancreatic islets. J. Clin. Endocrinol. Metab. 82, 2471–2475 (1997).

    CAS  Google Scholar 

  69. Beattie, G.M., Butler, C. & Hayek, A. Morphology and function of cultured human fetal pancreatic cells transplanted into athymic mice: a longitudinal study. Cell Transplant. 3, 421–425 (1994).

    Article  CAS  Google Scholar 

  70. Kanai, Y. et al. Identification of two Sox17 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis. J. Cell Biol. 133, 667–681 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Matthias Hebrok, Mike German and Alberto Hayek for critical review of the manuscript; Alberto Hayek for total RNA from 11- and 14-week fetal human pancreas; and Michael McCaffery at John Hopkins University for performing the EM analyses. SU5402 was a gift from A. Terskikh, mouse anti-NKX6-1 (F55A12) and rabbit anti-NKX6-1 were gifts from Ole Madsen, and rabbit anti-NGN3 was a gift from Mike German. The CyT203 and CyT49 cell lines were derived with partial funding from the Juvenile Diabetes Research Foundation.

Author information

Authors and Affiliations

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

    Kevin A D'Amour, Anne G Bang, Susan Eliazer, Olivia G Kelly, Alan D Agulnick, Nora G Smart, Mark A Moorman, Evert Kroon, Melissa K Carpenter & Emmanuel E Baetge

Authors
  1. Kevin A D'Amour
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Anne G Bang
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Susan Eliazer
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Olivia G Kelly
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Alan D Agulnick
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Nora G Smart
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Mark A Moorman
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Evert Kroon
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Melissa K Carpenter
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. Emmanuel E Baetge
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence to Emmanuel E Baetge.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Endocrine differentiation from hES cells is critically dependent on the DE lineage. (PDF 578 kb)

Supplementary Fig. 2a

Endocrine differentiation from hES cells via DE but not neural or extra-embryonic lineages. (PDF 529 kb)

Supplementary Fig. 2b

Endocrine differentiation from hES cells via DE but not neural or extra-embryonic lineages. (PDF 523 kb)

Supplementary Fig. 3

Comparison of mRNA expression levels in hES-derived cells to fetal and adult human pancreas. (PDF 469 kb)

Supplementary Fig. 4

Amylase expressing cells are intermingled with endocrine cells. (PDF 1213 kb)

Supplementary Fig. 5

Endocrine clusters stain with dithizone. (PDF 1054 kb)

Supplementary Fig. 6

Numerous secretory granules in hES cell-derived cells. (PDF 825 kb)

Supplementary Fig. 7

Differentiation of 5 different hES cell lines to insulin-expressing cells. (PDF 541 kb)

Supplementary Fig. 8

MAFB and insulin are co-expressed in hES cell-derived cells and human fetal pancreas. (PDF 1199 kb)

Supplementary Table 1

Real-time PCR primers. (PDF 199 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

D'Amour, K., Bang, A., Eliazer, S. et al. Production of pancreatic hormone–expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24, 1392–1401 (2006). https://doi.org/10.1038/nbt1259

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1259

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing