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Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells

Abstract

Using a flow cytometry–based screen of commercial antibodies, we have identified cell-surface markers for the separation of pancreatic cell types derived from human embryonic stem (hES) cells. We show enrichment of pancreatic endoderm cells using CD142 and of endocrine cells using CD200 and CD318. After transplantation into mice, enriched pancreatic endoderm cells give rise to all the pancreatic lineages, including functional insulin-producing cells, demonstrating that they are pancreatic progenitors. In contrast, implanted, enriched polyhormonal endocrine cells principally give rise to glucagon cells. These antibodies will aid investigations that use pancreatic cells generated from pluripotent stem cells to study diabetes and pancreas biology.

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Figure 1: Cell-surface markers for endocrine and PE cells in hES cell–derived pancreatic cultures.
Figure 2: FACS isolation of CD142 and CD200 cell subsets from hES cell–derived pancreatic cultures.
Figure 3: CD142, CD200 and CD318 immuno-magnetic cell separations from hES cell–derived pancreatic cultures.
Figure 4: Immunofluorescence analyses of transplants of unenriched or CD318-enriched endocrine cells.
Figure 5: Immunofluorescence and functional analyses of transplants of unenriched or CD142-enriched PE cells.

References

  1. 1

    Guo, T. & Hebrok, M. Stem cells to pancreatic beta-cells: new sources for diabetes cell therapy. Endocr. Rev. 30, 214–227 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Van Hoof, D., D'Amour, K.A. & German, M.S. Derivation of insulin-producing cells from human embryonic stem cells. Stem Cell Res. (Amst.) 3, 73–87 (2009).

    Article  CAS  Google Scholar 

  3. 3

    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 

  4. 4

    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 

  5. 5

    Sugiyama, T. & Kim, S.K. Fluorescence-activated cell sorting purification of pancreatic progenitor cells. Diabetes Obes. Metab. 10 (suppl. 4), 179–185 (2008).

    Article  PubMed  Google Scholar 

  6. 6

    McKnight, K.D., Wang, P. & Kim, S.K. Deconstructing pancreas development to reconstruct human islets from pluripotent stem cells. Cell Stem Cell 6, 300–308 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Winkler, H. & Fischer-Colbrie, R. The chromogranins A and B: the first 25 years and future perspectives. Neuroscience 49, 497–528 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Pan, F.C. & Wright, C. Pancreas organogenesis: from bud to plexus to gland. Dev. Dyn. 240, 530–565 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. 9

    Murray, H.E., Paget, M.B. & Downing, R. Preservation of glucose responsiveness in human islets maintained in a rotational cell culture system. Mol. Cell. Endocrinol. 238, 39–49 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. 10

    Gao, R., Ustinov, J., Korsgren, O. & Otonkoski, T. In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells. Diabetologia 48, 2296–2304 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. 11

    Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. 12

    Miyazawa, Y. et al. CUB domain-containing protein 1, a prognostic factor for human pancreatic cancers, promotes cell migration and extracellular matrix degradation. Cancer Res. 70, 5136–5146 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. 13

    Uhlen, M. et al. Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 28, 1248–1250 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. 14

    Luther, T. et al. Tissue factor expression during human and mouse development. Am. J. Pathol. 149, 101–113 (1996).

    PubMed  PubMed Central  CAS  Google Scholar 

  15. 15

    Moberg, L. et al. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet 360, 2039–2045 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. 16

    Beuneu, C. et al. Human pancreatic duct cells exert tissue factor-dependent procoagulant activity: relevance to islet transplantation. Diabetes 53, 1407–1411 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. 17

    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).

    PubMed  CAS  Google Scholar 

  18. 18

    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 

  19. 19

    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).

    PubMed  CAS  Google Scholar 

  20. 20

    Herrera, P.L. Adult insulin- and glucagon-producing cells differentiate from two independent cell lineages. Development 127, 2317–2322 (2000).

    PubMed  CAS  Google Scholar 

  21. 21

    Wilson, M.E., Kalamaras, J.A. & German, M.S. Expression pattern of IAPP and prohormone convertase 1/3 reveals a distinctive set of endocrine cells in the embryonic pancreas. Mech. Dev. 115, 171–176 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. 22

    Rezania, A. et al. Production of functional glucagon-secreting alpha cells from human embryonic stem cells. Diabetes 60, 239–247 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. 23

    Collombat, P. et al. The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells. Cell 138, 449–462 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Thorel, F. et al. Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss. Nature 464, 1149–1154 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Chung, C.H., Hao, E., Piran, R., Keinan, E. & Levine, F. Pancreatic beta-cell neogenesis by direct conversion from mature alpha-cells. Stem Cells 28, 1630–1638 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. 26

    Hald, J. et al. Generation and characterization of Ptf1a antiserum and localization of Ptf1a in relation to Nkx6.1 and Pdx1 during the earliest stages of mouse pancreas development. J. Histochem. Cytochem. 56, 587–595 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Braam, S.R., Nauw, R., Ward-van Oostwaard, D., Mummery, C. & Passier, R. Inhibition of ROCK improves survival of human embryonic stem cell-derived cardiomyocytes after dissociation. Ann. NY Acad. Sci. 1188, 52–57 (2010).

    Article  CAS  PubMed  Google Scholar 

  28. 28

    Koyanagi, M. et al. Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors. J. Neurosci. Res. 86, 270–280 (2008).

    Article  CAS  PubMed  Google Scholar 

  29. 29

    Krawetz, R.J., Li, X. & Rancourt, D.E. Human embryonic stem cells: caught between a ROCK inhibitor and a hard place. Bioessays 31, 336–343 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. 30

    Jiang, W. et al. CD24: a novel surface marker for PDX1-positive pancreatic progenitors derived from human embryonic stem cells. Stem Cells 29, 609–617 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. 31

    Dorrell, C. et al. Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers. Stem Cell Res. (Amst.) 1, 183–194 (2008).

    Article  CAS  Google Scholar 

  32. 32

    Sugiyama, T., Rodriguez, R.T., McLean, G.W. & Kim, S.K. Conserved markers of fetal pancreatic epithelium permit prospective isolation of islet progenitor cells by FACS. Proc. Natl. Acad. Sci. USA 104, 175–180 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. 33

    Hori, Y., Fukumoto, M. & Kuroda, Y. Enrichment of putative pancreatic progenitor cells from mice by sorting for prominin1 (CD133) and platelet-derived growth factor receptor beta. Stem Cells 26, 2912–2920 (2008).

    Article  CAS  PubMed  Google Scholar 

  34. 34

    Nostro, M.C. et al. Stage-specific signaling through TGFbeta family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells. Development 138, 861–871 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank O. Madsen (Hagedorn Research Institute), C. Wright (Vanderbilt University), J. Johnson (UT Southwestern Medical Center) and BD Biosciences for providing antibodies and A. Elefanty and E. Stanley (Monash University) for providing the MEL1 hES cell line. The CyT203 and CyT49 hES cell lines were derived with partial funding from the Juvenile Diabetes Research Foundation.

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O.G.K. and A.G.B. wrote the paper. O.G.K. and A.G.B. designed, directed and interpreted experiments with intellectual contributions from E.E.B., M.M., L.A.M., E.K., K.A.D., K.K. and M.K.C. The antibody screen was proposed by A.G.B. and carried out by O.G.K., M.Y.C. and M.M. K.A.D. suggested the Y-27632 compound. M.M. developed and performed the flow cytometry assays and analyses with assistance from M.Y.C. and K.G.R. O.G.K., A.G.B., M.Y.C. and T.M.O. performed the cell culture experiments and immuno-magnetic cell separations. M.Y.C. and O.G.K. performed qPCR and immunofluorescence analyses of in vitro material. L.A.M., E.K. and M.R. executed the in vivo experiments, including transplantations and C-peptide assays. K.K. performed the histological and immunofluorescence analyses of implanted grafts.

Corresponding author

Correspondence to Olivia G Kelly.

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The authors are employees or former employees of ViaCyte (formerly Novocell).

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Supplementary Table 1 and Supplementary Figures 1–9 (PDF 2938 kb)

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Kelly, O., Chan, M., Martinson, L. et al. Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells. Nat Biotechnol 29, 750–756 (2011). https://doi.org/10.1038/nbt.1931

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