Characterization Of Immortalized Mesenchymal Stem Cells Derived From Foetal Pocrine Pancreas And Their Differentiation Into Insulin Producing Cells

Following cardiovascular disease and cancer, Diabetes have been the third largest chronic diseases of high incidence. It will also cause a variety of complications, such as eye diseases, kidney diseases, nervous system diseases, cardiovascular diseases and diabetic hyperosmolar syndrome. The usage of porcine islet cells is currently viewed as the most promising alternative, as there is a plentiful supply of porcine islet cells; moreover, porcine and human insulins are highly conserved, and porcine normal physiological glucose levels are similar to those in humans. However, pancreatic stem cells (PSCs) are rare and have a finite proliferative lifespan, culminating in permanent population growth arrest–known as replicative senescence, resulting in inability to multiply, and to phenotypic instability.In this study, we isolated islet and established an immortalized mesenchymal stem cell (MSC) line derived from foetal porcine pancreas, by transfecting human telomerase reverse transcriptase (hTERT) and called these immortalized pancreatic mesenchymal stem cells (iPMSCs). Biological characteristics of the cells were studied to determine whether the correlation with the characteristics of pancreatic stem cells and provide whether the unlimited resources for islet replacement therapy.1. Study on the isolation and biological properties of immortalized pancreatic mesenchymal stem cells derived from foetal porcine pancreas (iPMSCs)In this study, we isolated MSCs from 2.5 to 3 months old porcine fetal pancreas by a suspend-to-adhere culture method. And we established an immortalized mesenchymal stem cells (MSC) line derived from foetal porcine pancreas, by transfecting hTERT. After passage, the cells remained a strong proliferative activity (more than 100 generations). The biological characteristics of the cells were identified by cell growth curve, flow cytometry analysis of surface antigens, immuno?uorescence/immunocytochemistry staining, RT-PCR, tumor detection and differentiation into neural, cardiac and germ-like cells in vitro. Nude mice were monitored and no tumour outgrowths wer eobserved for up to 2 months after injection with iPMSCs.2. Differentiation of iPMSCs into insulin-secreting cells in vitro We used a two-step protocol of differentiation in vitro. First, iPMSCs were cultured inRPMI1640 ?B27medium supplemented with 100 mmol/L nicotinamide, 100 ng/mL ActivinA, 20 ng/mL EGF and 20 ng/mL bFGF for 4 days. Cells were then transferred to ultra-low attachment plates and cultured in the same medium for 3 days. Another 7 days in the same medium supplemented with 10 nmol/L Exendin4, 4 nmol/L Betacellulin (BTC) and without EGF or bFGF. Medium was changed every 3 days. IPMSCs were differentiated into islet-like clusters with this protocol. The induced clusters were DTZ, PDX1, Insulin and C-peptide positive. With low and high concentrations of glucose in a static assay, cell clusters derived from iPMSCs were capable of secreting insulin and C-peptide in response to glucose stimulation. Injecting with STZ, diabetic mice were successfully established. And the induced cells transplanted into diabetic mice, which could reverse hyperglycaemia after transplantation into diabetic mice, but time that blood glucose levels were reduced were short (2 w). Then iPMSCs were transplanted into mouse testes, immunological staining revealed that the cells could spontaneously differentiate into germ cells in vivo and spontaneously differentiated into insulin-secreting cells in high glucose conditions.3. GSK3 inhibitor-BIO regulates the proliferation of iPMSCsAfter adding BIO in vitro, we preliminarily concluded that BIO could promote iPMSCs proliferation with Wnt/β-catenin signaling pathway by immunofluorescence staining, QRT-PCR, Western-blotting, luciferase activity analysis, BrdU proliferation assay and apoptosis analysis (Tunel staining), however, we did not find the related roles of BIO onβcell differentiation by immunostaining, QRT-PCR assay and glucose-stimulated insulin release and insulin content analysis. These results suggest that BIO plays a key role in the regulation of cell proliferation and maintenance of the undifferentiated state of iPMSCs.These results indicated that iPMSCs has strong proliferation and differentiation capacity in vitro, and can provide adequate cell resources for tissue engineering and regenerative medicine.