Endocrine precursor cells from mouse islets are not generated by epithelial-to-mesenchymal transition of mature beta cells - PubMed (original) (raw)

Endocrine precursor cells from mouse islets are not generated by epithelial-to-mesenchymal transition of mature beta cells

Russell A Morton et al. Mol Cell Endocrinol. 2007.

Abstract

We previously presented evidence that proliferative human islet precursor cells may be derived in vitro from adult islets by epithelial-to-mesenchymal transition (EMT) and show here that similar fibroblast-like cells can be derived from mouse islets. These mouse cell populations exhibited changes in gene expression consistent with EMT. Both C-peptide and insulin mRNAs were undetectable in expanded cultures of mouse islet-derived precursor cells (mIPCs). After expansion, mIPCs could be induced to migrate into clusters and differentiate into hormone-expressing islet-like aggregates. Although early morphological changes suggesting EMT were observed by time-lapse microscopy when green fluorescent protein-labeled beta cells were placed in culture, the expanded precursor cell population was not fluorescent. Using two mouse models in which beta cells were permanently made either to express alkaline phosphatase or to have a deleted M(3) muscarinic receptor, we provide evidence that mIPCs in long term culture are not derived from beta cells.

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Figures

Figure 1

Figure 1

Derivation of islet precursor cells from preparations of adult mouse islets. a) A phase contrast image of a mouse islet after 3 days of in vitro culture. b) A mouse islet after 9 days in vitro. Left: Differential interference contrast (DIC) image. Right: Confocal image (1 μm optical slice) after immunostaining with anti-C-peptides 1 and 2 and anti-glucagon antibodies. c) Phase contrast image of mesenchymal mouse islet-derived precursor cells (mIPCs) after 50 days in culture. d) Growth curves for 3 preparations of mouse mesenchymal cells. Ms01 and Ms02 were derived from normal mice and MIP-GFP01 was derived from a MIP-GFP transgenic mouse.

Figure 2

Figure 2

Differentiation of islet precursor cells from MIP-GFP mice. Micrographs depict clusters of passage 12 mesenchymal cells that had been expanded at least 4000-fold. Cells were cultured in differentiation medium for 7 days prior to fixation, embedding in paraffin and sectioning. Left: DIC image. Right: Confocal image (0.6 μm optical slice) after immunostaining with anti-C-peptides 1 and 2 and anti-glucagon antibodies.

Figure 3

Figure 3

Selected images from time-lapse microscopy during in vitro culture of MIP-GFP islets. Images of a single MIP-GFP islet were acquired from days 3 through 7 at 15 minute intervals. Single images from each day from day 3 (d3) through day 7 (d7) are presented. Left: Phase contrast; Right: GFP epifluorescence.

Figure 4

Figure 4

PCR analysis of genomic DNA from M3 muscarinic receptor knockout mouse. Islets were prepared from floxed M3 muscarinic receptor mutant mice (15) expressing Cre recombinase from the rat insulin promoter. Genomic DNA was isolated from a freshly isolated pancreas digest (Pancreas), from islet clusters after 2 days in RPMI media (Day 2), from adherent cells after 12 days (Day 12) and two days later (Day 14). PCR results using primer pairs 1/2 (290 bp), 3/4 (720 bp) and 5/6 (704 bp) are shown. The 290 bp product from primer pair 1/2 identifies the M3 receptor locus after excision by Cre recombinase and is present in genomic DNA from pancreas and after 2 days in culture but is lost by days 12 and 14 in the resulting mesenchymal cell cultures.

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