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Int. J. Mol. Sci. 2017, 18(11), 2234;

FGF-2b and h-PL Transform Duct and Non-Endocrine Human Pancreatic Cells into Endocrine Insulin Secreting Cells by Modulating Differentiating Genes

Department of Systems Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
Department of Human Sciences and Quality of Life Promotion, San Raffaele Roma Open University, 00166 Rome, Italy
Agenzia regionale per la protezione ambientale (ARPA) Lazio, Sezione di Roma, 00173 Rome, Italy
Endocrinology and Metabolism of Transplantation, Azienda Ospedaliero-Universitaria (A.O.U.) Pisana, 56126 Pisa, Italy
Department of Medicine, Surgery and Neuroscience, University of Siena, 53100 Siena, Italy
These authors contributed equally to this work.
Author to whom correspondence should be addressed.
Received: 3 August 2017 / Revised: 13 October 2017 / Accepted: 16 October 2017 / Published: 25 October 2017
(This article belongs to the Special Issue Genome Editing 2018)
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Background: 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
Keywords: pancreatic β cells; cellular differentiation; insulin release; regenerative medicine; diabetes mellitus pancreatic β cells; cellular differentiation; insulin release; regenerative medicine; diabetes mellitus

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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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.

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