title: In vitro evaluation of different protocols for the induction of mesenchymal stem cells to insulin-producing cells.
abstract: Stem cells therapy is a new promising approach for diabetes mellitus (DM) treatment, but the insulin secretion rate in differentiated cells is low when compared with pancreas beta cells embedded in Langerhans islets. In this study, we evaluated different protocols of insulin secretion to achieve the most appropriate protocol for in vitro insulin secretion. We differentiated human umbilical cord matrix-derived mesenchymal cells (hUCMs) into insulin-producing cell (IPC) by the aim of three previously reported protocols and a modified protocol. The insulin content was analyzed through gene expression and immunocytochemistry (IHC). Dithizone (DTZ) staining was done for identification of islet-like structures. Insulin and C peptide secretion was measured by chemiluminesence immunoassay (CLIA) and enzyme immunoassay (EIA) as well. Reverse transcription-PCR (RT-PCR) showed efficient expression of insulin genes in all the study groups. IHC analysis showed higher expression of insulin and proinsulin proteins in the modified protocol. DTZ staining exhibited variable islet-like clusters in the different protocols except control. This finding was confirmed by the higher response to glucose challenge test in this group. A modified protocol using an intermediate step that makes the cells vulnerable to nestin production in combination with inducing agent results in the higher differentiation of stem cells into insulin-producing cells and more insulin secretion in vitro.
authors.affiliation: Department of Anatomy, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran.|Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran.|Afzal Research Institute, Kerman, Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Seyedi
authors.affiliation: Stem Cell Research Lab., Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Alireza
authors.initials: A
authors.lastname: Farsinejad
authors.affiliation: Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Mojgan
authors.initials: M
authors.lastname: Moshrefi
authors.affiliation: Department of Anatomy, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran. nnematollahi@kmu.ac.ir.|Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran. nnematollahi@kmu.ac.ir.|Physiology Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran. nnematollahi@kmu.ac.ir.
authors.forename: Seyed Noureddin
authors.initials: SN
authors.lastname: Nematollahi-Mahani
mesh_terms: D002454:Cell Differentiation; D017077:Culture Media, Conditioned; D005434:Flow Cytometry; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D008163:Luminescent Measurements; D059630:Mesenchymal Stem Cells / Q000187:drug effects* / Q000502:physiology; D011384:Proinsulin / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017077:Culture Media, Conditioned; D007328:Insulin; D011384:Proinsulin
keywords: Differentiation protocol; Human umbilical cord matrix-derived mesenchymal cells (hUCMs); Insulin-producing cell (IPC); Nestin