The results support the notion that INGAP peptide is a potent inducer of islet cell neogenesis. We have previously described the in vitro induction of human adult islet-to-duct cell transdifferentiation in which the cells of the resulting duct-like cystic structures exhibit a primitive epithelial morphology.
We hypothesize that some or all these cells may exhibit developmental plasticity and could thus be used as an expandable pool of islet cell progenitors.
Islet neogenesis associated protein (INGAP) is a mediator of in vivo islet cell neogenesis from pancreatic duct epithelial cells in several species.
The aim of the present study was to induce islet cell neogenesis in vitro by treating these putative progenitor cells with INGAP peptide, a pentadecapeptide of the native protein.
Freshly isolated adult human pancreatic islet cells (>95% pure) were induced to differentiate into duct-like epithelial cells in a collagen gel supplemented with a defined basal medium containing cholera toxin (100ng/ml) (CT). As shown by immunohistochemistry (IHC), islet-to-duct transformation was characterized by the loss of expression of PDX-1 and islet cell hormones, and the appearance, in all cells, of the duct-epithelial cell marker CK-19. After 10 days, cultures were either supplemented with INGAP peptide (250 ng/ml) (Group 1) or maintained in basal medium (Group 2) for an additional four days. Data was compiled in triplicate from each of 4 separate islet isolations representing 4 different human donors.
Following addition of INGAP peptide, as observed under the inverted microscope, 34% of duct-like cystic structures acquired a solid islet-like appearance. In contrast, 0% of the cystic structures in Group 2 underwent this change in morphology. By IHC, 88% of the islet-like structures in Group 1 were positive for PDX-1; 83% were positive for insulin; 79% were positive both PDX-1 and insulin and all were negative for CK-19. At the cellular level, 25±5% of all cells became immunoreactive for PDX-1 (vs 8±2% in Gr. 2, p<0.01); 21±5% became positive for insulin (vs 0% in Gr. 2), 4±1% became positive for glucagon (vs 0% in Gr. 2); 1±0% became positive for somatostatin (vs 0% in Gr.2); and 19.6±6% of cells expressed both PDX-1 and insulin (vs 0% in Gr. 2).
These results support the notion that INGAP peptide is a potent inducer of islet cell neogenesis from a putative adult islet progenitor cell, and that the induction of endocrine differentiation is mediated by PDX-1, a transcription factor known to be required for pancreatic Beta-cell development. ENDO 2002 (OR30-2)