Aims/hypothesis An immunohistochemical and genomic analysis of human pancreatic development from 9-23 weeks of fetal age was undertaken to provide a comparative analysis of human and murine islet development. Methods Human fetal pancreases obtained at gestational ages 9-23 weeks were processed in parallel for immunohistochemistry and gene expression profiling by Affymetrix microarrays.

Cell adhesion molecules (CAMs) are important mediators of cell–cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell–cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.

Widespread application of -cell replacement strategies for diabetes is dependent upon the availability of an unlimited supply of cells exhibiting appropriate glucose-responsive insulin secretion. Therefore, a great deal of effort has been focused on understanding the factors that control -cell growth. Previously, we found that human -cell-enriched islet cultures can be stimulated to proliferate, but expansion was limited by growth arrest after 10-15 cell divisions. Here, we have investigated the mechanism behind the growth arrest. Our studies, including analyses of the expression of senescence-associated -galactosidase, p16INK4a levels, and telomere lengths, indicate that cellular senescence is responsible for limiting the number of cell divisions that human -cells can undergo. The senescent phenotype was not prevented by retroviral transduction of the hTERT gene, although telomerase activity was induced. These results have implications for the use of primary human islet cells in cell transplantation therapies for diabetes.

Isolation of endocrine cell precursors from the human fetal pancreas will be important to the study of islet cytodifferentiation and eventually for islet transplantation in insulin-dependent diabetes. These precursor cells, from which all four islet endocrine cell types arise, are present within fetal pancreatic ductal epithelium. After enzymatic digestion and culture of the fetal pancreas, we obtained cell clusters resembling islets, but with a high content of undifferentiated cells. Histochemical staining revealed very high acid beta-galactosidase activity in over 70% of cells within the clusters. After transplantation into athymic nude mice, the islet-like cell clusters gave rise to tissue rich in differentiated endocrine cells, but low in beta-galactosidase activity. The histochemical finding of high acid beta-galactosidase activity in endocrine precursor cells was confirmed by direct measurement of lysosomal enzyme activities. In addition, we found that the expression of acid beta-galactosidase was developmentally regulated, peaking at 18-24 weeks gestation and declining to low levels in adult islets. Using a fluorogenic beta-galactosidase substrate, we were able to isolate a subpopulation of cells high in acid beta-galactosidase activity using fluorescence-activated flow cytometry. Evidence identifying these cells as potential islet cell precursors includes, besides the transplantation experiments, the colocalization in vitro of tyrosine hydroxylase, a marker of embryonic islet cells. Thus, our results indicate that high acid beta-galactosidase activity serves as a marker for a population of fetal pancreatic cells with the potential to differentiate and grow into mature pancreatic endocrine cells.

Aims and hypothesis. Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family with a high degree of specificity for epithelial cells in vitro and in vivo. Our aim was to study the effect of KGF on beta-cell growth and differentiation on islet-like cell clusters derived from human fetal pancreas. Methods. We investigated the effects of KGF, in vitro, on beta-cell differentiation from undifferentiated pancreatic precursor cells and in vivo after transplantating human fetal pancreatic cells into athymic rats treated with KGF. Results. Treatment of islet-like cell clusters with KGF in vitro did not change the number of insulin producing cells, as measured by the measurement of insulin content or DNA. The in vivo treatment of recipient rats with KGF increased the number of beta cells within the grafts 8 weeks after transplantation. At this time, glucose-stimulated insulin secretion was evaluated by glucose stimulation tests in rats bearing the transplants. Measurements of human C-peptide concentrations after glucose challenge showed that the newly differentiated beta cells in the KGF-treated group were functionally competent as opposed to the control group, where the graft failed to release insulin appropriately. Conclusion/interpretation. These findings suggest that in vivo, KGF is capable of inducing human fetal beta-cell expansion. The growth promoting effect of KGF on beta cells occurred mainly through the activation of ductal cell proliferation and their subsequent differentiation into beta cells. [Diabetologia (2003) 46:822-829]

lsolalion 01 Ihe endocr~ne cell precursors lrom the tiurnan felai pancreas is Important lor the undersland~ng 01 Islet cytod~fferent~al~on The progcnltor cells, lrom wtlich all lour islet endocr~ne cell types arlse, are presenl w~t h~n tile ep~thelium of the fetal H~stociietnical staiiilng revealed vety hlgh acid 1%-galactosidasc act~vily in most cells wllI!ln tllc clusters. Alter Irany>larital~oii i~ito altlyrnlc nude [nice. Ilis clusters gave rlse to tissue rich in d~fferentiated endocrine cells. The histochern~cal find~ng of high ac~d p galactosidase actlv~ly was confirmed by d~recl measurement of lysosamal enzyme activities, It1 add~l~oli, we lound that the expresston ol a c~d 1%-galactosidase was deveiopmentally regulaled, peaking at 18-24 weeks gestation and declining to low levels In adult islets Using a fluorogenic p.galactosidase substrate, we were able lo enrich for a subpopulation of cells hlgh In a c~d (3-galactos~dase act~vity with llow cytomelty. Evidence ~denlilyng these cells as pote~itial islct cell precursors tncluded, besides the transplanlation experiments. Ihe coiocal~zat~on In 'v~tro of tyros~ne hydroxylase, a known marker of emblyon~c ~slct celis Thus, our r~s~i l l s indicale lhat hlgh acd Il-galactostdase activity serves as a lnarkcr lo enricli poptilaltons 01 endocrine cell precursors.B r e n d e l , R. A l e j a n d r o and J. N e r u p , S t e n o D i a b e t e s C e n t e r , 2820 G e n t o f t e , D e n m a r k a n d D i a b e t e s R e s e a r c h I n s t i t u t e , U n i v e r s i t y o f Miami, M i a m i , FL 33101, U S A T h e c y t o k i n e IL-1 0 inhibits rodent 0-cell function i n a time-a n d dose-dependent m a n n e r a n d h a s b e e n ~m p l i c a t e d a s effector molecule ~n t h e I D D M pathogenesis. In isolated rat islets IL-1 toxicity t o O-cells is dependent o n generation o f free nitric oxide radicals. Nicotinamide (NA). a free radical s c a v e n g e r a n d inhibitor o f p o l y -A D P -r i b o s e synthetase p r e v e n t s m a c r o p h a g e m e d i a t e d islet cell destruction a n d a u t o i m m u n e diabetes i n the NOD m o u s e , a n d N A s h o w s promising preventive effects i n prediabetic h u m a n s . H e r e w e studied I L -I sensitivity and N A protection of isolated h u m a n islets using a completely tntra-species i n vitro system: A 2 4 hr h u m a n islet e x p o s u r e t o authentic recombinant h u m a n IL-I 0 A 6 h r h u m a n islet I L -I exposure already leads t o a 3 0 % d e c r e a s e of insulin accumulation while i n rat islets functional i n h~b i t i o n i s s e e n only after 2 4 h r s o f IL-1 exposure . I n conclusion isolated h u m a n islets are m o s t sensitive t o IL-1 i n d u c e d O-cell suppression a n d N A protects h u m a n islet function against the IL-1 attack. The development of 0verweig;lt has recently been recognized as a common finding especially in adolecents with IDDM. However, both normal longitudinal growth and the prevention of overweight are major goals in pediatric diabetology. We therefore inves...