Insulin-expressing beta cells, found in pancreatic islets, are capable of generating more beta cells even in the adult. We show that fibroblast-like cells derived from adult human islets donated postmortem proliferate readily in vitro. These mesenchymal-type cells, which exhibit no hormone expression, can then be induced to differentiate into hormone-expressing islet-like cell aggregates, which reestablishes the epithelial character typical of islet cells. Immunohistochemistry, in situ hybridization, and messenger RNA measurements in single cells and cell populations establish the transition of epithelial cells within islets to mesenchymal cells in culture and then to insulin-expressing epithelial cells.

No abstract

No abstract

No abstract

Development of the endocrine pancreas includes a series of early events wherein precursor cells cluster, that is migrate to form cell aggregates, which subsequently differentiate into islets of Langerhans. We show that PANC-1 cells, a human pancreatic cell line, differentiates into hormone-producing islet-like cell aggregates after exposure to a defined serum-free medium. These cells were used to provide the following evidence that fibroblast growth factor (FGF)2 is a paracrine chemoattractant during PANC-1 cell clustering: (i) FGF2 is secreted and remains bound to the extracellular matrix from where it may diffuse to form chemoattractive gradients; (ii) a subset of cells expresses FGF receptors (FGFRs) -1, -2, -3 , and -4; (iii) inhibition of FGFR tyrosine kinase inhibits cell clustering; and (iv) FGF2 neutralizing antibody inhibits clustering. In addition, adult human islet-derived precursor cells, which cluster and differentiate in a manner similar to PANC-1 cells, also secrete FGF2 and express FGFRs. We conclude that FGF2, acting as a paracrine chemoattractant, stimulates clustering of precursor cells, an early step leading to islet-like cell aggregate formation. Similar processes may occur during development of the islet of Langerhans in humans.migration ͉ paracrine ͉ aggregation ͉ differentiation

The mammalian pancreas is known to show a remarkable degree of regenerative ability. Several studies until now have demonstrated that the mammalian pancreas can regenerate in normal as well as diabetic conditions. These studies illustrate that pancreatic transcription factors that are seen to be expressed in a temporal fashion during development are re-expressed during regeneration. The only known exception to this is Neurogenin3 (NGN3). Though NGN3 protein, which marks all the pro-endocrine cells during development, is not seen during mouse pancreas regeneration, functional neo-islets are generated by 4 weeks after 70% pancreatectomy. We observed that pancreatic transcription factors upstream of ngn3 showed similar gene expression patterns during development and regeneration. However, gene transcripts of transcription factors immediately downstream of ngn3 (neuroD and nkx2.2) did not show such similarities in expression. Since NGN3 protein was not detected at any time point during regeneration, we reasoned that post-transcriptional silencing of ngn3 by microRNAs may be a possible mechanism. We carried out microRNA analysis of 283 known and validated mouse microRNAs during different stages of pancreatic development and regeneration and identified that 4 microRNAs; miR-15a, miR-15b, miR-16 and miR-195, which can potentially bind to ngn3 transcript, are expressed at least 200-fold higher in the regenerating mouse pancreas as compared to embryonic day (e) 10.5 or e 16.5 developing mouse pancreas. Inhibition of these miRNAs in regenerating pancreatic cells using anti-sense miRNA-specific inhibitors, induces expression of NGN3 and its downstream players: neuroD and nkx2.2. Similarly, overexpression of miRNAs targeting ngn3 during pancreas development shows reduction in the number of hormone-producing cells. It appears that during pancreatic regeneration in mice, increased expression of these microRNAs allows endocrine regeneration via an alternate pathway that does not involve NGN3 protein. Our studies on microRNA profiling of developing and regenerating pancreas provide us with better understanding of mechanisms that regulate post-natal islet neogenesis.

Epithelial-to-mesenchymal transition is a phenomenon necessary for embryonic development and also seen during certain pathological conditions.  We show here for the first time that reduction in miR-30 family microRNAs, is responsible for mesenchymal transition of primary cultures of human pancreatic epithelial cells.  We found that miR-30 family microRNAs target mesenchymal gene transcripts and maintain them in a translationally inactive state.  Forced depletion using miR-30 family specific anti-miRs leads to mesenchymal transition while ectopic overexpression maintains the epithelial phenotype.  We also show that miR-30 family microRNAs increase in abundance during differentiation of pancreatic islet-derived mesenchymal cells into hormone-producing islet-like cell aggregates.  Our studies in human adult diseased pancreas also demonstrate that miR-30 family microRNAs are expressed at lower abundance in fibrotic lesions during pancreatitis.  Together, our data confirm that miR-30 family microRNAs form a part of the regulatory signaling events involved in cellular response of pancreatic epithelial cells during mesenchymal transition.

No abstract