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

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.

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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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Correspondence to Raewyn M Seaberg.

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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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