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:

A small molecule that directs differentiation of human ESCs into the pancreatic lineage

Nature Chemical Biology volume 5, pages 258–265 (2009)Cite this article

Abstract

Stepwise differentiation from embryonic stem cells (ESCs) to functional insulin-secreting beta cells will identify key steps in beta-cell development and may yet prove useful for transplantation therapy for diabetics. An essential step in this schema is the generation of pancreatic progenitors—cells that express Pdx1 and produce all the cell types of the pancreas. High-content chemical screening identified a small molecule, (−)-indolactam V, that induces differentiation of a substantial number of Pdx1-expressing cells from human ESCs. The Pdx1-expressing cells express other pancreatic markers and contribute to endocrine, exocrine and duct cells, in vitro and in vivo. Further analyses showed that (−)-indolactam V works specifically at one stage of pancreatic development, inducing pancreatic progenitors from definitive endoderm. This study describes a chemical screening platform to investigate human ESC differentiation and demonstrates the generation of a cell population that is a key milepost on the path to making beta cells.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: High-content screen and the characterization of a hit compound.
Figure 2: ILV-treated hESC-derived cells express multiple pancreatic markers.
Figure 3: ILV-treated HUES 8-E cells can further differentiate into multiple pancreatic lineages, in vitro and in vivo.
Figure 4: ILV functions specifically at one stage of pancreatic development: the induction of pancreatic progenitors from GTE through PKC activation.

Similar content being viewed by others

Article 01 December 2022

Zixuan Zhao, Xinyi Chen, … Hanry Yu

References

  1. Anonymous. Regenerative Medicine. National Institutes of Health http://stemcells.nih.gov/info/scireport/2006report.htm (2006).

  2. Lumelsky, N. et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 292, 1389–1394 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Soria, B. In-vitro differentiation of pancreatic beta-cells. Differentiation 68, 205–219 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. 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).

    PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  8. D'Amour, K.A. et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat. Biotechnol. 24, 1392–1401 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. McLean, A.B. et al. Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed. Stem Cells 25, 29–38 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Jiang, W. et al. In vitro derivation of functional insulin-producing cells from human embryonic stem cells. Cell Res. 17, 333–344 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Ku, H.T. et al. Committing embryonic stem cells to early endocrine pancreas in vitro. Stem Cells 22, 1205–1217 (2004).

    Article  PubMed  Google Scholar 

  12. Gu, G., Brown, J.R. & Melton, D.A. Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis. Mech. Dev. 120, 35–43 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. 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  PubMed  Google Scholar 

  14. Offield, M.F. et al. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122, 983–995 (1996).

    CAS  PubMed  Google Scholar 

  15. 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  PubMed  Google Scholar 

  16. Tiso, N., Filippi, A., Pauls, S., Bortolussi, M. & Argenton, F. BMP signalling regulates anteroposterior endoderm patterning in zebrafish. Mech. Dev. 118, 29–37 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Roy, S., Qiao, T., Wolff, C. & Ingham, P.W. Hedgehog signaling pathway is essential for pancreas specification in the zebrafish embryo. Curr. Biol. 11, 1358–1363 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. diIorio, P.J., Moss, J.B., Sbrogna, J.L., Karlstrom, R.O. & Moss, L.G. Sonic hedgehog is required early in pancreatic islet development. Dev. Biol. 244, 75–84 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. 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  PubMed  Google Scholar 

  20. 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  PubMed  Google Scholar 

  21. Kroon, E. et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat. Biotechnol. 26, 443–452 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Ding, S. & Schultz, P.G. A role for chemistry in stem cell biology. Nat. Biotechnol. 22, 833–840 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Chen, S. et al. Self-renewal of embryonic stem cells by a small molecule. Proc. Natl. Acad. Sci. USA 103, 17266–17271 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Osafune, K. et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotechnol. 26, 313–315 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Frandsen, U., Porneki, A.D., Floridon, C., Abdallah, B.M. & Kassem, M. Activin B mediated induction of Pdx1 in human embryonic stem cell derived embryoid bodies. Biochem. Biophys. Res. Commun. 362, 568–574 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Kim, S.K. & Melton, D.A. Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor. Proc. Natl. Acad. Sci. USA 95, 13036–13041 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 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  PubMed  Google Scholar 

  28. Kawaguchi, Y. et al. The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nat. Genet. 32, 128–134 (2002).

    Article  CAS  PubMed  Google Scholar 

  29. Beck, F., Erler, T., Russell, A. & James, R. Expression of Cdx-2 in the mouse embryo and placenta: possible role in patterning of the extra-embryonic membranes. Dev. Dyn. 204, 219–227 (1995).

    Article  CAS  PubMed  Google Scholar 

  30. 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 

  31. Bendayan, M. & Ito, S. Immunohistochemical localization of exocrine enzymes in normal rat pancreas. J. Histochem. Cytochem. 27, 1029–1034 (1979).

    Article  CAS  PubMed  Google Scholar 

  32. Osafune, K. et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotechnol. 26, 313–315 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Korsgren, O., Jansson, L., Eizirik, D. & Andersson, A. Functional and morphological differentiation of fetal porcine islet-like cell clusters after transplantation into nude mice. Diabetologia 34, 379–386 (1991).

    Article  CAS  PubMed  Google Scholar 

  34. Movassat, J., Beattie, G.M., Lopez, A.D. & Hayek, A. Exendin 4 up-regulates expression of PDX 1 and hastens differentiation and maturation of human fetal pancreatic cells. J. Clin. Endocrinol. Metab. 87, 4775–4781 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Seymour, P.A. et al. SOX9 is required for maintenance of the pancreatic progenitor cell pool. Proc. Natl. Acad. Sci. USA 104, 1865–1870 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Sherwood, R.I. et al. Prospective isolation and global gene expression analysis of definitive and visceral endoderm. Dev. Biol. 304, 541–555 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Hirota, M. et al. Synthetic analogues (indolactams) of (−)-indolactam-V are new congeners of the teleocidin class of tumor promoters. Jpn. J. Cancer Res. 78, 577–582 (1987).

    CAS  PubMed  Google Scholar 

  38. Heikkila, J. & Akerman, K.E. (−)-Indolactam V activates protein kinase C and induces changes in muscarinic receptor functions in SH-SY5Y human neuroblastoma cells. Biochem. Biophys. Res. Commun. 162, 1207–1213 (1989).

    Article  CAS  PubMed  Google Scholar 

  39. Griner, E.M. & Kazanietz, M.G. Protein kinase C and other diacylglycerol effectors in cancer. Nat. Rev. Cancer 7, 281–294 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Gschwendt, M. et al. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett. 392, 77–80 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. Martiny-Baron, G. et al. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J. Biol. Chem. 268, 9194–9197 (1993).

    CAS  PubMed  Google Scholar 

  42. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26, 101–106 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Park, I.H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Ochoa, W.F. et al. Retinoic acid binds to the C2-domain of protein kinase C(alpha). Biochemistry 42, 8774–8779 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Radominska-Pandya, A. et al. Direct interaction of all-trans-retinoic acid with protein kinase C (PKC). Implications for PKC signaling and cancer therapy. J. Biol. Chem. 275, 22324–22330 (2000).

    Article  CAS  PubMed  Google Scholar 

  48. Rosewicz, S., Brembeck, F., Kaiser, A., Marschall, Z.V. & Riecken, E.O. Differential growth regulation by all-trans retinoic acid is determined by protein kinase C alpha in human pancreatic carcinoma cells. Endocrinology 137, 3340–3347 (1996).

    Article  CAS  PubMed  Google Scholar 

  49. Bouzinba-Segard, H., Fan, X.T., Perderiset, M. & Castagna, M. Synergy between phorbol esters and retinoic acid in inducing protein kinase C activation. Biochem. Biophys. Res. Commun. 204, 112–119 (1994).

    Article  CAS  PubMed  Google Scholar 

  50. Mailhos, C., Howard, M.K. & Latchman, D.S. A common pathway mediates retinoic acid and PMA-dependent programmed cell death (apoptosis) of neuronal cells. Brain Res. 644, 7–12 (1994).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

D.M. and S.L.S. are investigators of the Howard Hughes Medical Institute. S.C. is supported by the Howard Hughes Medical Institute and Juvenile Diabetes Research Foundation. R.M. is supported by the Howard Hughes Medical Institute. L.F.P. is supported by the American Gastroenterological Association and the National Pancreas Foundation. The authors thank D. Huangfu for thoughtful discussion, H. Ngo for assistance with the high-content screen, S. Huang for help with NMR analysis and the Beta Cell Biology Consortium for providing antibodies.

Author information

Author notes

  1. Malgorzata Borowiak and Julia L Fox: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Cambridge, Massachusetts, USA

    Shuibing Chen, Malgorzata Borowiak, Julia L Fox, René Maehr, Kenji Osafune, Lance Davidow, Kelvin Lam, Lee L Rubin & Douglas Melton

  2. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    Shuibing Chen, René Maehr & Douglas Melton

  3. Department of Chemistry and Chemical Biology, Harvard University, Howard Hughes Medical Institute, Cambridge, Massachusetts, USA

    Lee F Peng & Stuart L Schreiber

Authors
  1. Shuibing Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Malgorzata Borowiak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Julia L Fox
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. René Maehr
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kenji Osafune
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lance Davidow
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kelvin Lam
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lee F Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Stuart L Schreiber
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Lee L Rubin
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Douglas Melton
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.C., D.M. and S.L.S. designed the experiments, analyzed data and wrote the manuscript; S.C., M.B., R.M. and K.O. performed the experiments; J.L.F., L.F.P., S.L.S. and L.L.R. provided the chemical library; and S.C., L.D., K.L. and L.L.R. performed the high-content screen.

Corresponding author

Correspondence to Douglas Melton.

Ethics declarations

Competing interests

The authors have filed a patent on the ILV molecule described in the paper.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13, Supplementary Table 1 and Supplementary Methods (PDF 2306 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, S., Borowiak, M., Fox, J. et al. A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat Chem Biol 5, 258–265 (2009). https://doi.org/10.1038/nchembio.154

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.154

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