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Int. J. Mol. Sci. 2017, 18(11), 2234; doi:10.3390/ijms18112234

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

1
Department of Systems Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
2
Department of Human Sciences and Quality of Life Promotion, San Raffaele Roma Open University, 00166 Rome, Italy
3
Agenzia regionale per la protezione ambientale (ARPA) Lazio, Sezione di Roma, 00173 Rome, Italy
4
Endocrinology and Metabolism of Transplantation, Azienda Ospedaliero-Universitaria (A.O.U.) Pisana, 56126 Pisa, Italy
5
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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Abstract

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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    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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