In vitro trans-differentiation of bone marrow stromal cells into islet-like clusters by dimethyl sulphoxide

Abstract：BACKGROUND: Recent findings suggest that bone marrow stromal cells (MSCs) have the capacity to differentiate into neurons induced by dimethyl sulphoxide (DMSO). So we think whether MSCs can trans-differentiate to nestin positive precurosor cells, then to endocrine cells of the pancreas.OBJECTIVE: To investigate the possibility of differentiating functional insulin-producing cells from MSCs induced by DMSO. DESIGN: Randomized and controlled trails for cells in vitro.SETTING: Department of Histology and Embryology, Medical College of Hebei North University. MATERIALS: This study was performed in the Cell Culture Room of Department of Histology and Embryology, Hebei North University from May 2006 to July 2007. Ten SPF Wistar rats of 6-8 weeks age without sex constraint weighing 180-230 g, were purchased from the Center of Experimental Animal Academy of Military Medical Sciences (license: SCXK-armed forces 2002-001). Fetal bovine serum was Gibco products, Trizol and RT-PCR (A3500) kit were purchased from Promega; DMSO from ChemicalReagent Beijing Co., Ltd.; Taq enzyme and DNA marker DL2000 from Tianwei Biotech Beijing Co., Ltd.; mice anti-human insulin monoclonal antibody, rabbit anti-human glucagon and nestin polyclonal antibody from Zhongshan Golden Bridge Biotechnology Co., Ltd.; The polyclonal antibodies had well cross-reaction with the rat's tissue. So they could be used to tissues of human, rats or other mammals. SABC kit was provided by Boster Biotechnology Co., Ltd.; radioimmunoassay kit by Linco Co., Ltd.METHODS: The bone marrow cells woe cultured in H-DMEM supplemented with 0.1 volume fraction of FBS. After 60-minute incubation, non-adherent cells were collected. The cells were re-plated in serum-free DMEM medium at a cell density of 109/cm2 in the presence of 10 mL/L DMSO. The cells were then cultured in DMEM supplemented with 10 mL/L DMSO for 3 days followed by high (25 mmol/L) glucose DMEM media supplemented with 0.1 volume fraction of FBS for 7 additional days. They were plated in plastic six well plates on slide.MAIN OUTCOME MEASURES: The morphology were observed and diphenyl thiocarbazone (DTZ) staining. Immuno-activity was detected using the cytochemical method for the protein expression, such as nestin, insulin, glucagons and somatostatin. The endocrine gene expressions of induced clusters were detected by RT-PCR. Measurement of Insulin content and secretion were detected by immunoassay.RESULTS: The MSC cells woe typically fibrocyte-like, grape-like coherent dnstered cells. They were observed under experiment group alter the 3rd day after DMSO induction, which were similar to pancreatic islet cells. DTZ staining of the cell aggregates were positive as the natural pancreatic islets, Immunocytochernistry also confirmed that these aggregates were positive for nestin on the 1st day after DMSO induction, and positive for insulin, glucagon on the 10th day after DMSO; while the control groups were negatively stained for the characteristics of endocrine. RT-PCR results showed the insulin 1, insulin 2, glucagon and somatostatin geae expressions were on the 7th day after DMSO induction. The aggregates have good reactions to glucose, the secretion of insulin content respectively 5.56 and 24.5 μ U/mL under low glucose (3.3 mmol/L) and high glucose (16.7 mmol/L) conditions.CONCLUSION: The MSCs can be induced to differentiate into pancreatic endocrine hormone-producing cells in serum-flee with DMSO.