Differentiation of COPAS-sorted non-endocrine pancreatic cells into insulin-positive cells in the mouse

R. Kikugawa, H. Katsuta, T. Akashi, S. Yatoh, G. C. Weir, A. Sharma, S. Bonner-Weir

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Aims/hypothesis: The regenerative process in the pancreas is of particular interest, since insulin-producing beta cells are lost in diabetes. Differentiation of new beta cells from pancreatic non-endocrine cells has been reported in vivo and in vitro, a finding that implies the existence of pancreatic stem/progenitor cells. However, while tissue-specific stem cells are well documented in skin, intestine and testis, pancreatic stem cells have been elusive. We hypothesised that pancreatic stem/progenitor cells within the non-endocrine fraction could be a source of new islets in vitro. Methods: To test if there were such cells within the pancreas, we generated pancreatic cell aggregates from tissue remaining after islet isolation from mouse insulin promoter 1-green fluorescent protein (MIP-GFP) mice. To eliminate any contamination of insulin-positive cells, we deleted all GFP-positive aggregates using COPAS Select and cultured with Matrigel. Immunohistochemistry, quantitative real-time PCR and single-cell nested RT-PCR were performed to confirm formation of insulin-producing cells. Results: The GFP-negative cells were expanded as monolayers and then differentiated into three-dimensional cystic structures. After 1 week of culture, GFP-positive cells were found as clusters or single cells. By quantitative real-time PCR, no insulin mRNA was detected immediately after COPAS sorting, but after differentiation insulin mRNA of the whole preparation was 1.91∈±∈0.31% that of purified MIP-GFP beta cells. All GFP-positive cells expressed insulin 1; most expressed insulin 2, pancreas duodenum homeobox-1 and cytokeratin 19 by single cell nested RT-PCR. Conclusions/interpretation: Our data support the concept that within the exocrine (acinar and ductal) pancreas of the adult mouse there are cells that can give rise to insulin-positive cells in vitro.

Original languageEnglish
Pages (from-to)645-652
Number of pages8
JournalDiabetologia
Volume52
Issue number4
DOIs
Publication statusPublished - Apr 2009

All Science Journal Classification (ASJC) codes

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

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