FGF-2b and h-PL Transform Duct and Non-Endocrine Human Pancreatic Cells into Endocrine Insulin Secreting Cells by Modulating Differentiating Genes
AbstractBackground: Diabetes mellitus (DM) is a multifactorial disease orphan of a cure. Regenerative medicine has been proposed as novel strategy for DM therapy. Human fibroblast growth factor (FGF)-2b controls β-cell clusters via autocrine action, and human placental lactogen (hPL)-A increases functional β-cells. We hypothesized whether FGF-2b/hPL-A treatment induces β-cell differentiation from ductal/non-endocrine precursor(s) by modulating specific genes expression. Methods: Human pancreatic ductal-cells (PANC-1) and non-endocrine pancreatic cells were treated with FGF-2b plus hPL-A at 500 ng/mL. Cytofluorimetry and Immunofluorescence have been performed to detect expression of endocrine, ductal and acinar markers. Bromodeoxyuridine incorporation and annexin-V quantified cells proliferation and apoptosis. Insulin secretion was assessed by RIA kit, and electron microscopy analyzed islet-like clusters. Results: Increase in PANC-1 duct cells de-differentiation into islet-like aggregates was observed after FGF-2b/hPL-A treatment showing ultrastructure typical of islets-aggregates. These clusters, after stimulation with FGF-2b/hPL-A, had significant (p < 0.05) increase in insulin, C-peptide, pancreatic and duodenal homeobox 1 (PDX-1), Nkx2.2, Nkx6.1, somatostatin, glucagon, and glucose transporter 2 (Glut-2), compared with control cells. Markers of PANC-1 (Cytokeratin-19, MUC-1, CA19-9) were decreased (p < 0.05). These aggregates after treatment with FGF-2b/hPL-A significantly reduced levels of apoptosis. Conclusions: FGF-2b and hPL-A are promising candidates for regenerative therapy in DM by inducing de-differentiation of stem cells modulating pivotal endocrine genes. View Full-Text
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Description: Supplemental Figure 1. Confocal microscopy analysis of PDX-1 internalization. Islet-like aggregates obtained after 96h of treatment of PANC-1 cells with FGF-2b plus hPL-A (A, B) and stimulated with glucose (20mM for 1 hours) (B), were disaggregated to form single cell suspensions. Cells were then centrifuged with a cytospin on polilysine-coated slides and immediately fixed by paraformaldehyde 4%. After permeabilization, cells were stained for PDX-1 (green) while nuclei were blue-stained by Hoechst (A, B). Confocal microscopy analysis showed that treatment with FGF-2b plus hPL-A induced an increase of PDX-1 staining in the nucleus, after stimulation with glucose (B) that induces PDX-1 internalization. Space bar = 20 µm.
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Donadel, G.; Pastore, D.; Della-Morte, D.; Capuani, B.; Lombardo, M.F.; Pacifici, F.; Bugliani, M.; Grieco, F.A.; Marchetti, P.; Lauro, D. FGF-2b and h-PL Transform Duct and Non-Endocrine Human Pancreatic Cells into Endocrine Insulin Secreting Cells by Modulating Differentiating Genes. Int. J. Mol. Sci. 2017, 18, 2234.
Donadel G, Pastore D, Della-Morte D, Capuani B, Lombardo MF, Pacifici F, Bugliani M, Grieco FA, Marchetti P, Lauro D. FGF-2b and h-PL Transform Duct and Non-Endocrine Human Pancreatic Cells into Endocrine Insulin Secreting Cells by Modulating Differentiating Genes. International Journal of Molecular Sciences. 2017; 18(11):2234.Chicago/Turabian Style
Donadel, Giulia; Pastore, Donatella; Della-Morte, David; Capuani, Barbara; Lombardo, Marco F.; Pacifici, Francesca; Bugliani, Marco; Grieco, Fabio A.; Marchetti, Piero; Lauro, Davide. 2017. "FGF-2b and h-PL Transform Duct and Non-Endocrine Human Pancreatic Cells into Endocrine Insulin Secreting Cells by Modulating Differentiating Genes." Int. J. Mol. Sci. 18, no. 11: 2234.
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