Aims/hypothesis: Transplantation of insulinproducing beta cells from donors can cure diabetes, but they are available in insufficient quantities. In this study, we investigated the possibility of generating insulinproducing cells from adult rat exocrine cells cultured in the presence of growth factors. Methods: Rat exocrine pancreatic cells were isolated and treated in vitro with epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF). Analysis was performed by immunocytochemistry, DNA measurement and radioimmunoassay. Cells were transplanted to alloxan-treated (70 mg/kg) nude mice and glycaemia was monitored for 21 days. Nephrectomy was performed on day 15. Results: In a 3-day culture period, addition of LIF plus EGF to the medium resulted in an 11-fold increase of the beta cell mass. This could not be attributed to the very low mitotic activity of contaminating beta cells. Furthermore, when contaminating beta cells were initially destroyed with alloxan, this effect was even more pronounced. The newly formed cells secreted insulin in response to glucose and were immunoreactive for C-peptide-I, Pdx-1 and GLUT-2, which are characteristics of mature beta cells. Electron microscopy showed that they also contained insulinimmunoreactive secretory granules. Some insulin-positive cells were immunoreactive for amylase and cytokeratin-20, or were binucleated, which are characteristics of exocrine cells. The cells were able to restore normoglycaemia when transplanted to alloxan-diabetic mice, and hyperglycaemia recurred upon removal of the graft. Conclusions/interpretation: Our study shows that functional beta cells can be generated from exocrine tissue by transdifferentiation and thereby may offer a new perspective for beta cell therapy.

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When pancreatic tissue is injured after duct obstruction, acinoductal metaplasia is observed. Similar metaplastic changes occur when exocrine pancreatic cells are isolated and cultured. We demonstrate that under these experimental conditions the exocrine acinar cells lose their differentiated characteristics: expression of the acinar transcription factors p48/ Ptf1␣ and Mist1 is decreased or lost, whereas expression of the embryonic transcription factor Pdx1 is increased. The receptors Notch1 and Notch2, members of the DSL family of Notch ligands, and the target genes in the Notch-signaling pathway Hes1, Hey1, and Hey2 become strongly up-regulated. We noted also reduced expression of Sel1L, a Notch repressor that is normally highly expressed in exocrine pancreas. Stimulation of Notch by its ligand Jagged1 diminished the proliferation of cultured metaplastic exocrine cells. Chemical inhibition of Notch signaling resulted in increased proliferation and induction of the cell-cycle regulator p21Cip1 . This effect seems to be Hes1-independent and mainly coincides with decreased Hey1 and Hey2 mRNA expression. In conclusion , we demonstrate that during acinoductal metaplasia the Notch-signaling pathway is activated concomitantly with changes in transcription factor expression of pancreatic acinar cells. In addition, we show that Notch signaling is implicated in the suppression of proliferation of these metaplastic exocrine cells. The latter may be important in protection from neoplastic transformation.

Aims/hypothesis. We investigated the expression and function of netrin-1, a diffusible laminin-like protein known to regulate neuronal-cell migration in the pancreas. We questioned whether this factor regulates migration of pancreatic epithelial cells and whether this could be involved in islet neogenesis. Methods. We studied fetal and adult rat pancreas wherein duct ligation induced islet neogenesis. Netrin-1 expression was analysed by RT-PCR, western blot and immunohistochemistry. In vitro cell migration was measured with a human pancreatic duct cell line (CAPAN-2) and with fetal porcine islet cells. We also studied the expression of two netrin-receptors, neogenin and deleted in colorectal cancer. Results. We found a transient expression of netrin-1 mRNA and protein in fetal pancreas from E15 to E18, and in adult pancreas after duct ligation. In normal adult pancreas there was very little netrin-1 expression. Netrin-1 expression was observed both in endocrine and exocrine cells. At the immunohistochemical level, it was expressed by islet cells during tissue regeneration. We could show that netrin-1 increases the migration of fetal islet cells and of a ductal cell line, mainly via a chemokinetic effect. From the two wellestablished netrin receptors, DCC and neogenin, we only found neogenin to be expressed in the pancreas. Neogenin expression coincided with the period of netrin-1 up-regulation. Conclusion/interpretation. Netrin-1 is involved in pancreatic morphogenesis and tissue remodelling and plays a role in the regulation of duct-cell and fetalislet cell migration. This can be of importance in islet regeneration, where migration of islet precursors takes place. [Diabetologia (2003) 46:926-933]

Aims/hypothesis: It was recently reported that culturing adult exocrine cells in the presence of epidermal growth factor and leukaemia inhibitory factor (LIF) resulted in their transdifferentiation into endocrine beta cells. The aim of this study was to examine the expression and function of LIF in the pancreas. Materials and methods: We studied the expression of LIF and its receptor components, LIF-receptor-β and gp130, by immunohistochemistry, western blotting and RT-PCR in normal rat pancreas, pancreas with duct ligation-induced islet neogenesis, and in pancreatic cell cultures. Isolated duct fragments were cultured in the presence of LIF and a janus kinase 2 (JAK2) inhibitor. Results: LIF was detected by immunohistochemistry, western blot and RT-PCR in the ducts of the normal pancreas. Both LIF-receptor-β and gp130 were detected by RT-PCR in the pancreas. Immunostaining revealed gp130 exclusively in the ducts and centro-acinar cells. After duct ligation-induced tissue injury, upregulation of LIF and its receptor occurred in rat pancreas. Metaplastic exocrine cells also started to express LIF and this was increased after alloxan treatment. Signalling via LIF-receptor-β/gp130 involves the JAK/signal transducer and activator of transcription (STAT) pathway. LIF induced increased activation of STAT3 in pancreatic cells. In isolated duct fragments, addition of LIF resulted in a significant increase in duct cell proliferation, while a specific inhibitor of the JAK/STAT signalling pathway inhibited proliferation. Conclusion/interpretation: Our observations show that LIF and its receptor are expressed in cells from pancreatic ducts. The cytokine plays a role in pancreatic physiology, controls duct cell proliferation and is involved in repair processes following pancreatic injury.