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Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages

Nature Biotechnology volume 22, pages 1115–1124 (2004)Cite this article

Abstract

The clonal isolation of putative adult pancreatic precursors has been an elusive goal of researchers seeking to develop cell replacement strategies for diabetes. We report the clonal identification of multipotent precursor cells from the adult mouse pancreas. The application of a serum-free, colony-forming assay to pancreatic cells enabled the identification of precursors from pancreatic islet and ductal populations. These cells proliferate in vitro to form clonal colonies that coexpress neural and pancreatic precursor markers. Upon differentiation, individual clonal colonies produce distinct populations of neurons and glial cells, pancreatic endocrine β-, α- and δ-cells, and pancreatic exocrine and stellate cells. Moreover, the newly generated β-like cells demonstrate glucose-dependent Ca2+ responsiveness and insulin release. Pancreas colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. These cells represent a previously unidentified adult intrinsic pancreatic precursor population and are a promising candidate for cell-based therapeutic strategies.

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Figure 1: PMP colonies are formed from progenitors present in adult pancreatic islet and duct cell isolates, and express markers characteristic of both neural and pancreatic precursors.
Figure 2: PMP colonies generate all three major neural cell lineages.
Figure 3: Progeny from two distinct embryonic primary germ layers are generated by single, clonally derived PMPs that are present in islet and ductal cell isolates.
Figure 4: Insulin+ cells generated de novo from PMPs demonstrate glucose-stimulated Ca2+ responses and glucose-stimulated insulin release.
Figure 5: PMP colonies generate multiple islet endocrine subtypes and exocrine cells.
Figure 6: PMPs are not general endodermal or mesodermal precursors, nor are they ES cell–like stem cells or neural crest precursors.

References

  1. Lechner, A. & Habener, J.F. Stem/progenitor cells from adult tissues: potential for the treatment of diabetes mellitus. Am. J. Physiol. Endocrinol. Metab. 284, E259–E266 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Ramiya, V.K. et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat. Med. 6, 278–282 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Hunziker, E. & Stein, M. Nestin-expressing cells in the pancreatic islets of Langerhans. Biochem. Biophys. Res. Commun. 271, 116–119 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Zulewski, H. et al. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 50, 521–533 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Abraham, E.J., Leech, C.A., Lin, J.C., Zulewski, H. & Habener, J.F. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. Endocrinology 143, 3152–3161 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Lendahl, U., Zimmerman, L.B. & McKay, R.D. CNS stem cells express a new class of intermediate filament protein. Cell 60, 585–595 (1990).

    Article  CAS  PubMed  Google Scholar 

  7. Weiss, S. et al. Is there a neural stem cell in the mammalian forebrain? Trends Neurosci. 19, 387–393 (1996).

    Article  CAS  PubMed  Google Scholar 

  8. Seaberg, R.M. & van der Kooy, D. Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. J. Neurosci. 22, 1784–1793 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hadjantonakis, A.-K., Gertsenstein, M., Ikawa, M., Okabe, M. & Nagy, A. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76, 79–90 (1998).

    Article  CAS  PubMed  Google Scholar 

  10. Morshead, C.M., Garcia, A.D., Sofroniew, M.V. & van der Kooy, D. The ablation of glial fibrillary acidic protein-positive cells from the adult central nervous system results in the loss of forebrain neural stem cells but not retinal stem cells. Eur. J. Neurosci. 18, 76–84 (2003).

    Article  PubMed  Google Scholar 

  11. Tropepe, V. et al. Retinal stem cells in the adult mammalian eye. Science 287, 2032–2036 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Li, H., Liu, H. & Heller, S. Pluripotent stem cells from the adult mouse inner ear. Nat. Med. 9, 1293–1299 (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. Guz, Y. et al. Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny. Development 121, 11–18 (1995).

    CAS  PubMed  Google Scholar 

  15. Kritzik, M.R. et al. PDX-1 and Msx-2 expression in the regenerating and developing pancreas. J. Endocrinol. 163, 523–530 (1999).

    Article  CAS  PubMed  Google Scholar 

  16. Zhang, Y.-Q. & Sarvetnick, N. Development of cell markers for the identification and expansion of islet progenitor cells. Diabetes Metab. Res. Rev. 19, 363–374 (2003).

    Article  PubMed  CAS  Google Scholar 

  17. Sommer, L., Ma, Q. & Anderson, D.J. Neurogenins, a novel family of atonal-related bHLH transcription factors, are putative mammalian neuronal determination genes that reveal progenitor cell heterogeneity in the developing CNS and PNS. Mol. Cell. Neurosci. 8, 221–241 (1996).

    Article  CAS  PubMed  Google Scholar 

  18. Bylund, M., Andersson, E., Novitch, B.G. & Muhr, J. Vertebrate neurogenesis is counteracted by Sox1–3 activity. Nat. Neurosci. 6, 1162–1168 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Guillemot, F. & Joyner, A.L. Dynamic expression of the murine achaete-scute homologue Mash-1 in the developing nervous system. Mech. Dev. 42, 171–185 (1993).

    Article  CAS  PubMed  Google Scholar 

  20. Liu, Y. & Rao, M. Oligodendrocytes, GRPs and MNOPs. Trends Neurosci. 26, 410–412 (2003).

    Article  PubMed  CAS  Google Scholar 

  21. 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 

  22. Kang, G. et al. Epac-selective camp analog 8-pCPT-2′-O-Me-cAMP as a stimulus for Ca2+-induced Ca2+ release and exoctyosis in pancreatic β-cells. J. Biol. Chem. 278, 8279–8285 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. MacDonald, P.E. et al. The multiple actions of GLP-1 on the process of glucose-stimulated insulin secretion. Diabetes 51, S434–S442 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Roe, M.W. et al. Expression and function of pancreatic beta-cell delayed rectifier K+ channels. Role in stimulus-secretion coupling. J. Biol. Chem. 271, 32241–32246 (1996).

    Article  CAS  PubMed  Google Scholar 

  25. Ahren, B., Karlsson, S. & Lindskog, S. Cholinergic regulation of the endocrine pancreas. Prog. Brain Res. 84, 209–218 (1990).

    Article  CAS  PubMed  Google Scholar 

  26. Lardon, J., Rooman, I. & Bouwens, L. Nestin expression in pancreatic stellate cells and angiogenic endothelial cells. Histochem. Cell Biol. 117, 535–540 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Seaberg, R.M. & van der Kooy, D. Stem and progenitor cells: the premature desertion of rigorous definitions. Trends Neurosci. 26, 125–131 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Mignone, J., Kukekov, V., Chiang, A.S., Steindler, D. & Enikolopov, G. Neural stem and progenitor cells in nestin-GFP transgenic mice. J. Comp. Neurol. 469, 311–324 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Sawamoto, K. et al. Generation of dopaminergic neurons in the adult brain from mesencephalic precursor cells labeled with a nestin-GFP transgene. J. Neurosci. 21, 3895–3903 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Weissman, I.L. Stem cells: Units of development, units of regeneration, and units in evolution. Cell 100, 157–168 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998).

    Article  CAS  PubMed  Google Scholar 

  32. Chambers, I. et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Gussoni, E. et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401, 390–394 (1999).

    CAS  PubMed  Google Scholar 

  34. Spangrude, G.J., Heimfeld, S. & Weissman, I.L. Purification and characterization of mouse hematopoietic stem cells. Science 241, 58–62 (1988).

    Article  CAS  PubMed  Google Scholar 

  35. Eftimie, R., Brenner, H. & Buonanno, A. Myogenin and MyoD join a family of skeletal muscle genes regulated by electrical activity. Proc. Natl. Acad. Sci. USA 88, 1349–1353 (1991).

    Article  CAS  PubMed  Google Scholar 

  36. Toma, J.G. et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat. Cell Biol. 3, 778–784 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. McKenzie, I. et al. Evidence that skin-derived precursor cells (skps) are an endogenous neural crest-derived precursor. Abstr. Soc. Neurosci. 28, 9 (2003).

    Google Scholar 

  38. Goulding, M.D., Chalepakis, G., Deutsch, U., Erselius, J.R. & Gruss, P. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J. 10, 1135–1147 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Stoetzel, C., Weber, B., Bourgeois, P., Bolcato Bellemin, A.L. & Perrin Schmitt, F. Dorso-ventral and rostro-caudal sequential expression of M-twist in the postimplantation murine embryo. Mech. Dev. 51, 251–263 (1995).

    Article  CAS  PubMed  Google Scholar 

  40. Honore, S.M., Aybar, M.J. & Mayor, R. Sox10 is required for the early development of the prospective neural crest in Xenopus embryos. Dev. Biol. 260, 79–96 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Lee, H.Y. et al. Instructive role of Wnt/beta-catenin in sensory fate specification in neural crest stem cells. Science 303, 1020–1023 (2004).

    Article  CAS  PubMed  Google Scholar 

  42. Aybar, M.J., Nieto, M.A. & Mayor, R. Snail precedes slug in the genetic cascade required for the specification and migration of the Xenopus neural crest. Development 130, 483–494 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Morrison, S.J., White, P.M., Zock, C. & Anderson, D.J. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell 96, 737–749 (1999).

    Article  CAS  PubMed  Google Scholar 

  44. Peterson, D.A., Leppert, J.T., Lee, K.-F. & Gage, F.H. Basal forebrain neuronal loss in mice lacking neurotrophin receptor p75. Science 277, 837–838 (1997).

    Article  CAS  PubMed  Google Scholar 

  45. Scharfmann, R. Neurotrophin and neurotrophin receptors in islet cells. Horm. Metab. Res. 29, 294–296 (1997).

    Article  CAS  PubMed  Google Scholar 

  46. Welsh, M. et al. The FRK/RAK tyrosine kinase participates in cytokine-induced islet cell toxicity. Biochem J. published online; doi: 10.1042/BJ20040285 (2004).

  47. Garcia-Ocana, A. et al. Transgenic overexpression of hepatocyte growth factor in the beta-cell markedly improves islet function and islet transplant outcomes in mice. Diabetes 50, 2752–2762 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. 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 

  49. 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  PubMed  Google Scholar 

  50. Persson-Sjogren, S., Zashihin, A. & Forsgren, S. Nerve cells associated with the endocrine pancreas in young mice: An ultrastructural analysis of the neuroinsular complex type I. Histochem. J. 33, 373–378 (2001).

    Article  CAS  PubMed  Google Scholar 

  51. Benveniste, P., Cantin, C., Hyam, D. & Iscove, N.N. Hematopoietic stem cells engraft in mice with absolute efficiency. Nat. Immunol. 4, 708–713 (2003).

    Article  CAS  PubMed  Google Scholar 

  52. Antonchuk, J., Sauvageau, G. & Humphries, R.K. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109, 39–45 (2002).

    Article  CAS  PubMed  Google Scholar 

  53. Jiang, Y. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41–49 (2002).

    Article  CAS  PubMed  Google Scholar 

  54. Clarke, D.L. et al. Generalized potential of adult neural stem cells. Science 288, 1660–1663 (2000).

    Article  CAS  PubMed  Google Scholar 

  55. Dor, Y., Brown, J., Martinez, O.I. & Melton, D. Adult pancreatic β-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429, 41–46 (2004).

    Article  CAS  PubMed  Google Scholar 

  56. Morshead, C.M., Benveniste, P., Iscove, N.N. & van der Kooy, D. Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations. Nat. Med. 8, 268–273 (2002).

    Article  CAS  PubMed  Google Scholar 

  57. Piper, K. et al. Beta-cell differentiation during human development does not rely on nestin-positive precursors: implications for stem cell-derived replacement therapy. Diabetologia 45, 1045–1047 (2002).

    Article  CAS  PubMed  Google Scholar 

  58. Humphrey, R.K. et al. Characterization and isolation of promoter-defined nestin-positive cells from the human fetal pancreas. Diabetes 52, 2519–2525 (2003).

    Article  CAS  PubMed  Google Scholar 

  59. Treutelaar, M.K. et al. Nestin-lineage cells contribute to the microvasculature but not endocrine cells of the islet. Diabetes 52, 2503–2512 (2003).

    Article  CAS  PubMed  Google Scholar 

  60. Delacour, A., Nepote, V., Trumpp, A. & Herrera, P.L. Nestin expression in pancreatic exocrine cell lineages. Mech. Dev. 121, 3–14 (2004).

    Article  CAS  PubMed  Google Scholar 

  61. Esni, F., Stoffers, D.A., Takeuchi, T. & Leach, S.D. Origin of exocrine pancreatic cells from nestin-positive precursors in the developing mouse pancreas. Mech. Dev. 121, 15–25 (2004).

    Article  CAS  PubMed  Google Scholar 

  62. Street, C.N. et al. Heterogenous expression of nestin in human pancreatic tissue precludes its use as an islet precursor marker. J. Endocrinol. 180, 213–225 (2004).

    Article  CAS  PubMed  Google Scholar 

  63. Klein, T. et al. Nestin is expressed in vascular endothelial cells in the adult human pancreas. J. Histochem. Cytochem. 51, 697–706 (2003).

    Article  CAS  PubMed  Google Scholar 

  64. Selander, L. & Edlund, H. Nestin is expressed in mesenchymal and not epithelial cells of the developing mouse pancreas. Mech. Dev. 113, 189–192 (2002).

    Article  CAS  PubMed  Google Scholar 

  65. Polesskaya, A., Seale, P. & Rudnicki, M.A. Wnt signaling induces the myogenic specification of resident CD45+ adult stem cells during muscle regeneration. Cell 113, 841–852 (2003).

    Article  CAS  PubMed  Google Scholar 

  66. Gotoh, M., Maki, J., Kiyoizumi, T., Satomi, S. & Monaco, A.P. An improved method for isolation of mouse pancreatic islets. Transplantation 40, 437–438 (1985).

    Article  CAS  PubMed  Google Scholar 

  67. Tropepe, V. et al. Distinct neural stem cells proliferate in response to EGF and FGF2 in the developing mouse telencephalon. Dev. Biol. 208, 166–188 (1999).

    Article  CAS  PubMed  Google Scholar 

  68. Smukler, S.R., Tang, L., Wheeler, M.B. & Salapatek, A.M.F. Exogenous nitric oxide and endogenous glucose-stimulated β-cell nitric oxide augment insulin release. Diabetes 51, 3450–3460 (2002).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank D. Dixon for excellent tissue isolation and purification, C. Smith for expert assistance with FACS analysis, A. Nagy for the gift of Oct4-GFP transgenic mice, M. Yamaguchi for the gift of nestin-GFP transgenic mice, M. Grompe for the gift of anti-FAH chicken polyclonal antibody and R.D.G. McKay for the gift of anti-nestin rabbit polyclonal antibody. This study was supported by the Canadian Stem Cell Network and the Canadian Institutes of Health Research.

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  1. Raewyn M Seaberg and Simon R Smukler: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medical Genetics and Microbiology, University of Toronto, Toronto, M5S 1A8, Canada

    Raewyn M Seaberg, Simon R Smukler & Derek van der Kooy

  2. Department of Physiology, University of British Columbia, Vancouver, V6T 1Z3, British Columbia, Canada

    Timothy J Kieffer

  3. Cold Spring Harbor Laboratory, Cold Spring Harbor, 11724, New York, USA

    Grigori Enikolopov

  4. Department of Physiology, University of Toronto, Toronto, M5S 1A8, Canada

    Zeenat Asghar & Michael B Wheeler

  5. Department of Surgery & Department of Physiology, University of Alberta, Edmonton, T6G 2N8, Alberta, Canada

    Gregory Korbutt

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Supplementary information

Supplementary Fig. 1

Differentiated PMP colonies contain β-cells that co-express Pax6 (red) and C-peptide (green). (PDF 253 kb)

Supplementary Fig. 2

PMPs are present in both nestin+ and nestin− cell fractions from both islet and ductal cell isolates, but all PMP colonies are nestin+ after 7 days in vitro. (PDF 209 kb)

Supplementary Table 1

Comparison of the gene expression profile of PMP colonies and brain-derived neurospheres by RT-PCR analysis, for both undifferentiated and differentiated conditions. (PDF 6 kb)

Supplementary Methods (PDF 13 kb)

Supplementary Notes (PDF 7 kb)

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Seaberg, R., Smukler, S., Kieffer, T. et al. Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages. Nat Biotechnol 22, 1115–1124 (2004). https://doi.org/10.1038/nbt1004

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