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Type 1 diabetes mellitus (T1D) is a chronic disease characterized by an autoimmune destruction of insulin-producing ß-pancreatic cells. Although many advances have been achieved in T1D treatment, current therapy strategies are often unable to maintain perfect control of glycemic levels. Several studies are searching for new and improved methodologies for expansion of ß-cell cultures in vitro to increase the supply of these cells for pancreatic islets replacement therapy. A promising approach consists of differentiation of stem cells into insulin-producing cells (IPCs) in sufficient number and functional status to be transplanted. Differentiation protocols have been designed using consecutive cytokines or signaling modulator treatments, at specific dosages, to activate or inhibit the main signaling pathways that control the differentiation of induced pluripotent stem cells (iPSCs) into pancreatic ß-cells. Here, we provide an overview of the current approaches and achievements in obtaining stem cell-derived ß-cells and the numerous challenges, which still need to be overcome to achieve this goal. Clinical translation of stem cells-derived ß-cells for efficient maintenance of long-term euglycemia remains a major issue. Therefore, research efforts have been directed to the final steps of in vitro differentiation, aiming at production of functional and mature ß-cells and integration of interdisciplinary fields to generate efficient cell therapy strategies capable of reversing the clinical outcome of T1D.

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Adipose-derived mesenchymal stem cells (ADSCs) are ideal sources for the treatment of diabetes, and the differentiation of ADSCs into insulin-producing cells (IPCs) through transfection of exogenous regulatory genes in vitro has been studied in depth. The differentiation of ADSCs is strictly regulated by a variety of transcription factors such as Pdx1, Ngn3, Pax4, Nkx2.2, and Sox9. However, whether these genes can coordinately regulate the differentiation of ADSCs into IPCs is still unknown. In this study, five multigene coexpressing adenovirus vectors (pAdTrack-Pdx1-Ngn3-AdEasy, pAdTrack-Pdx1-Ngn3-Sox9-AdEasy, pAdTrack-Pdx1-Ngn3-Pax4-Sox9-AdEasy, pAdTrack-Pdx1-Ngn3-Nkx2.2-Sox9-AdEasy, and pAdTrack-Pdx1-Ngn3-Nkx2.2-Pax4-AdEasy) were constructed, and then the stocks of the packaged adenoviruses were used to infect the canine ADSCs (cADSCs). Based on results of morphological observation, dithizone staining, sugar-stimulated insulin secretion test, cellular insulin immunofluorescence assays, and the detection of pancreatic ß-cell development-related genes in the induced cells, the best induction combination (pAdTrack-Pdx1-Ngn3-Nkx2.2-Pax4-AdEasy) was identified after comparative screening. This study provides a theoretical reference and an experimental basis for further research on stem cell replacement therapy for diabetes.

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BACKGROUND: Islet transplantation might be a logical strategy to restore insulin secretion for the treatment of diabetes, however, the scarcity of donors poses an obstacle for such a treatment. As an alternative islet source, differentiation of stem cells into insulin-producing cells (IPCs) has been tried. Many protocols have been developed to improve the efficiency of differentiation of stem cells into IPCs. In this study, we investigated whether glucosamine supplementation during differentiation of human adipose-derived stem cells (hADSCs) into IPCs can improve the insulin secretory function. METHODS: Glucosamine was added to the original differentiation medium at different stages of differentiation of hADSCs into IPCs for 12 days and insulin secretion was analyzed. RESULTS: Addition of glucosamine alone to the growth medium of hADSCs did not affect the differentiation of hADSCs to IPCs. Supplementation of the differentiation medium with glucosamine at a later stage (protocol G3) proved to have the greatest effect on IPC differentiation. Basal and glucose-stimulated insulin secretion (GSIS) was significantly increased and the expression of insulin and C-peptide was increased in differentiated IPCs as compared with that in differentiated IPCs using the conventional protocol (protocol C). In addition, the expression of beta-cell specific transcription factors such as pancreatic and duodenal homeobox1 (PDX1) and neurogenin 3 (NGN3) was also increased. Furthermore, the expression of genes related to insulin secretion, including synaptotagmin 4 (Syt4), glucokinase (Gck) and glucose transporter 2 (Glut2), was also increased. CONCLUSIONS: We conclude that glucosamine supplementation potentiates the differentiation of hADSCs into IPCs.

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BACKGROUND: Current approach for diabetes treatment remained several adverse events varied from gastrointestinal to life-threatening symptoms. Regenerative therapy regarding Edmonton protocol has been facing serious limitations involving protocol efficiency and safety. This led to the study for alternative insulin-producing cell (IPC) resource and transplantation platform. In this study, evaluation of encapsulated human dental pulp-derived stem cell (hDPSC)-derived IPCs by alginate (ALG) and pluronic F127-coated alginate (ALGPA) was performed. RESULTS: The results showed that ALG and ALGPA preserved hDPSC viability and allowed glucose and insulin diffusion in and out. ALG and ALGPA-encapsulated hDPSC-derived IPCs maintained viability for at least 336 h and sustained pancreatic endoderm marker (NGN3), pancreatic islet markers (NKX6.1, MAF-A, ISL-1, GLUT-2 and INSULIN), and intracellular pro-insulin and insulin expressions for at least 14 days. Functional analysis revealed a glucose-responsive C-peptide secretion of ALG- and ALGPA-encapsulated hDPSC-derived IPCs at 14 days post-encapsulation. CONCLUSION: ALG and ALGPA encapsulations efficiently preserved the viability and functionality of hDPSC-derived IPCs in vitro and could be the potential transplantation platform for further clinical application.

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Efficiency of the induction protocol is crucial for the generation of insulin-producing cells (IPCs) from human dental pulp stem cells (hDPSCs). Here, we established the integrative induction protocol by merging genetic manipulation technique with our previous published 3-step induction protocol aiming to enhance the pancreatic progenitor commitment and production yield. We found that the overexpression of PDX1 following with 3-step induction protocol were able to generate the 3-dimensional (3D) colony structure of pancreatic progenitors (PPs) with the beneficial trends of pancreatic endoderm commitment and production yield, while other protocols using the prolong maintenance of PDX1-overexpressed hDPSCs and the PDX1 overexpression after definitive endoderm induction were unable to generate and sustain the 3D structure of the colonies. Further Notch signaling manipulation by DAPT treatment showed lesser degree of positive effects on progenitor commitment and production yield. Although the generated PPs from the integrative protocol expressed pancreatic mRNA markers along with pro-insulin and insulin proteins, they still contained the defective glucose-responsive C-peptide secretion. Only basal secreted C-peptide level was observed. In summary, the integrative induction protocol potentially enhanced the PP generation with high colony production yield and could serve as an efficient platform for further hDPSC-derived IPC production and maturation.

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Insulin, a highly conserved peptide hormone, links nutrient availability to metabolism and growth in animals. In fed states insulin levels remain high and in animals that are food deprived insulin signalling drops. Here, we report that in Drosophila, feeding elicited by short periods of starvation is dependent on insulin signalling. The activity of insulin signalling pathway in the abdominal fatbody aids in feeding during short periods of starvation. A feedback regulatory signalling that involves cells that express the Drosophila hunger hormone short-neuropeptide-F (sNPF) and insulin-producing cells sustain the orexigenic function of insulin. Furthermore, the orexigenic phase of insulin activity aids in the efficient management of nutrient stores and survival of flies during starvation.

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Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into insulin-producing cells (IPCs). Bio-scaffolds derived from decellularized organs can act as a carrier for seed cells and may have broad applications in regenerative medicine. This study investigated the effect of native pancreatic stroma obtained from decellularized pancreas on the proliferation, migration and differentiation of BMSCs into IPCs, and explored the potential underlying molecular mechanism. The decellularized pancreas bio-scaffold was obtained by perfusion with Triton X-100/ammonium hydroxide, followed by digestion with a mixture of pepsin and hydrochloric acid to prepare the stroma solution. Islet-like cells were differentiated from BMSCs by a three-step induction method. The differences on the cytological behavior with or without stroma were evaluated by morphological observation, insulin release assay, qRT-PCR assay and western blot analysis. Our results showed that, stroma derived from decellularized pancreas could promote the proliferation and migration of BMSCs. Furthermore, the formation of IPCs could also be promoted, which possessed similar morphology to endogenous islets. During the induced differentiation process, the presence of stroma significantly increased the expression of insulin 1, insulin 2 and Pdx-1, as well as insulin release. This was accompanied by an increase in the phosphorylation of Akt and ERK in third stage cell clusters, which was prevented by the addition of the inhibitors PD98059 and LY294002, respectively. In summary, decellularized pancreatic stroma could promote the proliferation, migration and differentiation of BMSCs into IPCs, and this involved the activation of Akt and ERK signal pathways.

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To produce insulin-producing cells (IPCs) from bone marrow mesenchymal stem cells (BM-MSCs) using a simple and cost effective method. During the initial 7 days of three-dimensional (3D) culture, BM-MSCs were cultured on 1% agar or agarose to form multicellular spheroids. Spheroids and spheroid-derived single cells (SS and SSC, respectively) were cultured in the absence of any proteinaceous growth factor in a simple specific medium for a further 7 d. The insulin content of the differentiated cells was evaluated at the mRNA and protein levels. Furthermore, the expression of pancreatic beta cells-related genes other than INS as well as the in vitro responses of IPCs to different glucose concentrations were investigated. Cellular clusters generated on agar and SS conditions (agar+SS-IPCs) stained better with beta cell specific stains and were more reactive to serum-containing insulin reactive antibodies compared with agarose-SS-IPCs. Gene expression analysis revealed that in comparison to agarose + SS-IPCs, agar+SS-IPCs expressed significantly higher levels of INS-1, INS-2, PDX-1, NKX6.1, and XBP-1. Of interest, agar+SS-IPCs expressed 2215.3 ± 120.8-fold more INS-1 gene compared to BM-MSCs. The expression of ß-cell associated genes was also higher in agar+SS-IPCs compared to the agar+SSC-IPCs. Moreover, the expression of INS-1 gene was significantly higher in agar+SS-IPCs compared with agar+SSC-IPCs after culture in media with high concentration of glucose. Compared to the most expensive and time-consuming protocols, 3D culture of MSCs on agar followed by 2D culture of cellular clusters in a minimally supplemented high glucose media produced highly potent IPCs which may pay the way to the treatment of diabetic patients.

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BACKGROUND: The regulatory mechanism of insulin-producing cells (IPCs) differentiation from induced pluripotent stem cells (iPSCs) in vitro is very important in the phylogenetics of pancreatic islets, the molecular pathogenesis of diabetes, and the acquisition of high-quality pancreatic ß-cells derived from stem cells for cell therapy. METHODS: miPSCs were induced for IPCs differentiation. miRNA microarray assays were performed by using total RNA from our iPCs-derived IPCs containing undifferentiated iPSCs and iPSCs-derived IPCSs at day 4, day 14, and day 21 during step 3 to screen the differentially expressed miRNAs (DEmiRNAs) related to IPCs differentiation, and putative target genes of DEmiRNAs were predicted by bioinformatics analysis. miR-690 was selected for further research, and MPCs were transfected by miR-690-agomir to confirm whether it was involved in the regulation of IPCs differentiation in iPSCs. Quantitative Real-Time PCR (qRT-PCR), Western blotting, and immunostaining assays were performed to examine the pancreatic function of IPCs at mRNA and protein level respectively. Flow cytometry and ELISA were performed to detect differentiation efficiency and insulin content and secretion from iPSCs-derived IPCs in response to stimulation at different concentration of glucose. The targeting of the 3'-untranslated region of Sox9 by miR-690 was examined by luciferase assay. RESULTS: We found that miR-690 was expressed dynamically during IPCs differentiation according to the miRNA array results and that overexpression of miR-690 significantly impaired the maturation and insulinogenesis of IPCs derived from iPSCs both in vitro and in vivo. Bioinformatic prediction and mechanistic analysis revealed that miR-690 plays a pivotal role during the differentiation of IPCs by directly targeting the transcription factor sex-determining region Y (SRY)-box9. Furthermore, downstream experiments indicated that miR-690 is likely to act as an inactivated regulator of the Wnt signaling pathway in this process. CONCLUSIONS: We discovered a previously unknown interaction between miR-690 and sox9 but also revealed a new regulatory signaling pathway of the miR-690/Sox9 axis during iPSCs-induced IPCs differentiation.

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Tissue and stem cell encapsulation andtransplantation were considered as promising tools in the treatment of patients with diabetes mellitus. The aim of this study was to evaluate the effect of microfluidic encapsulation on the differentiation of trabecular meshwork mesenchymal stem cells (TM-MSC), into insulin-producing cells (IPCs) both in vitro and in vivo. The presence of differentiated cells in microfibers (three dimensional [3D]) and tissue culture plates (TCPS; two dimensional [2D]) culture was evaluated by detecting mRNA and protein expression of pancreatic islet-specific markers as well as measuring insulin release of cells in response to glucose challenges. Finally, semi-differentiated cells in microfibers (3D) and 2D cultures were used to control the glucose level in diabetic rats. The results of this study showed that MSCs differentiated in alginate microfibers (fabricated by microfluidic device) express more Pdx-1 mRNA (1.938-fold, p-value: 0.0425) and Insulin mRNA (2.841-fold, p-value: 0.0001) compared with those cultured on TCPS. Furthermore, cell encapsulation in microfluidic derived microfibers decreased the level of blood glucose in diabetic rats. The approach used in this study showed the possibility of alginate microfibers as a matrix for differentiation of TM-MSCs (as a new source) into IPCs. In addition, it could minimize different steps in stem cell differentiation, handling, and encapsulation, which lead to loss of an unlimited number of cells.

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Transplantation of stem cells using biocompatible nanofibrous scaffolds is a promising therapeutic method for treating Diabetic Mellitus. The aim of this study was to derive insulin-producing cells (IPCs) from conjunctiva-derived mesenchymal stem cell (CJMSCs) and to compare the functionality of differentiated IPCs in a three-dimensional (3D) culture with 2D. Furthermore, the effects of hydrophobicity of scaffold on IPCs differentiation were examined. Scanning electron microscopy (SEM), quantitative real times PCR (qPCR), Immunostaining and flow cytometry were used to analyze fabricated scaffold and the presence of IPCs. Functional maturity of differentiated cells was determined by measuring insulin release and the creation of IPCs was confirmed via gene and protein expression. In this study, the induced CJMSCs were morphologically similar to pancreatic islet-like cells. The expression of the islet-associated genes (glucagon, insulin and Pdx-1) and the insulin release (2.5-fold) in 3D-cultured cells was significantly higher than the 2D. The expression of IPCs genes was significantly higher in CJMSCs differentiated on plasma-treated nanofibers compared to those on untreated scaffolds. In conclusion, the results show that CJMSCs might be a new source for Diabetic Mellitus therapy and the nanofibrous scaffold could be used as a potential cell carrier for islet tissue engineering.

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BACKGROUND: Type 1 diabetes (T1D) is a multifactorial autoimmune disorder where pancreatic beta cells are lost before the clinical manifestations of the disease. Administration of mesenchymal stem cells (MSCs) or MSCs differentiated into insulin-producing cells (IPCs) have yielded limited success when used therapeutically. We have evaluated the immunoprophylactic potentials of precursors to insulin-producing cells (pIPCs) and IPCs in nonobese diabetic (NOD) mice to ask a basic question: do we need to differentiate MSCs into IPCs or will pIPCs suffice to attenuate autoimmune responses in T1D? METHODS: Bone marrow-derived MSCs from Balb/c mice were characterized following the International Society for Cellular Therapy (ISCT) guidelines. MSCs cultured in high-glucose media for 11 to 13 passages were characterized for the expression of pancreatic lineage genes using real-time polymerase chain reaction. Expression of the PDX1 gene in pIPCs was assessed using Western blot and fluorescence-activated cell sorting (FACS). Triple-positive MSCs were differentiated into IPCs using a three-step protocol after sorting them for cell surface markers, i.e. CD29, CD44, and SCA-1. Nonobese diabetic mice were administered pIPCs, IPCs, or phosphate-buffered saline (PBS) into the tail vein at weeks 9 or 10 and followed-up for 29-30 weeks for fasting blood glucose levels. Two consecutive blood sugar levels of more than 250 mg/dl were considered diabetic. RESULTS: MSCs grown in high-glucose media for 11 to 13 passages expressed genes of the pancreatic lineage such as PDX1, beta2, neurogenin, PAX4, Insulin, and glucagon. Furthermore, Western blot and FACS analysis for PDX-1, a transcription factor necessary for beta cell maturation, confirmed that these cells were precursors of insulin-producing cells (pIPCs). NOD mice administered with pIPCs were better protected from developing diabetes with a protective efficacy of 78.4% (p < 0.009); however, administration of IPCs gave protective efficacy of 55% at the end of 28-30 weeks. CONCLUSIONS: Precursors to insulin-producing cells seem to have better potential to arrest autoimmune response in type 1 diabetes when administered before the onset of the disease in NOD mice. When translated to humans, autologous mesenchymal stem cells grown in high-glucose media for 10 to 13 passages may have beneficial effects in individuals at high risk of developing type 1 diabetes.

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Ectopic expression of Pdx1 triggers rapid hepatocyte dedifferentiation by down-regulating liver-enriched transcription factors and liver-specific functional genes such as hepatic nuclear factor-1α (HNF1α), albumin, and AAT. However, the links between Pdx1 over-expression and hepatic gene down-regulation are incompletely understood. HNF1α and HNF4α are important transcription factors that establish and maintain the hepatocyte phenotype. The human HNF4α gene contains two promoters (P1 and P2) that drive expression of P1-(HNF4α 1-6) or P2-(HNF4α 7-9)-derived isoforms, which are used in different tissues and at different times during development. We hypothesized that the relative expression of HNF1α and HNF4α following ectopic Pdx1 expression may promote hepatic cell dedifferentiation and transdifferentiation toward pancreatic beta-cells. We produced lentiviruses expressing Pdx1, Pdx1-VP16, and Ngn3, along with dual-color reporter genes to indicate hepatic and pancreatic beta-cell phenotype changes. Using these PTF alone or in combinations, we demonstrated that Pdx1 not only activates specific beta-cell genes but down-regulates HNF1α. Pdx1-mediated reduction of HNF1α is accompanied by altered expression of its major activator, HNF4α isoforms, down-regulating hepatic genes ALB and AAT. Pdx1 up-regulates HNF4α via the P2 promoter. These P2-driven isoforms compete with P1-driven isoforms to suppress target gene transcription. In Huh7 cells, the AF-1 activation domain is more important for transactivation, whereas in INS1 cells, the F inhibitory domain is more important. The loss and gain of functional activity strongly suggests that Pdx1 plays a central role in reprogramming hepatocytes into beta-cells by suppressing the hepatic phenotype.

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Pancreatic islet transplantation has remained an effective therapy for type 1 diabetes since 2000. Its widespread use has been prohibited by the shortage of suitable donors. It is critical to explore an applicable alternative for ß-cell replacement. This study was performed to generate insulin-producing cells (IPCs) from pancreas-derived mesenchymal stem cells (pMSCs). pMSCs were isolated from discarded pancreatic tissue in the filter liquor during islet isolation procedure in mice and ex vivo expanded in culture. IPCs were induced by transfection of pancreas and duodenal transcription factor 1 (PDX-1) mRNA in vitro. Some islet characteristics were identified on pMSC-derived IPCs in mRNA and protein levels. Our results demonstrated that mouse pMSCs can be transdifferentiated into effective glucose-responsive insulin-producing cells through transfecting synthetic modified PDX-1 mRNA in vitro. The study of PDX-1 mRNA-induced pMSC reprogramming may pave the way toward the development of a novel ß-cell source for the treatment of diabetes.

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Objective To study the effects of micro-capsule on the secretion capacity of insulin-producing cells(IPCs). Methods Stem cells from originate mouse bone marrow mesenchymal were isolated,induced and purified. Rat pancre-atic extract(RPE)was extracted from pancreases of rats.BMMSCs were induced by rat pancreatic extract. The induced cells were randomly divided into micro-encapsulated group and non-micro-encapsulated group.The experiment of glu-cose stimulation was performed to detect the level of insulin,respectively, at different time points (1, 2, 3, 5, 10, 15, 20, 25, 30 day).Results The level of insulin secretion was increased after 1, 2, 3, 5 days of culture in micro-encapsu-lated IPCs and non-micro-encapsulated IPCs,but there were no significantly differences among the groups. The level of insulin secretion was declined in non-microencapsulated IPCs at 10 day,while there was no decreased in micro-en-capsulated IPCs until 20 days. Conclusion The micro-capsule can promote the effect duration of IPCs,which supports the function of IPCs.

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PURPOSE: Here we showed that human umbilical cord blood (hUCB)-derived cells, when cultured under defined conditions, generated insulin-producing cells (IPCs). METHODS: hUCB mononuclear cells (MNCs) were cultured in serum-free low (5.5 mM glucose) DMEM at a cell density of 3x10(6)/cm2 in the presence of 1% DMSO for 3 days followed by high (25 mM glucose) DMEM supplemented with 10% FBS for 7 additional days. They were plated in plastic six well plates on slide coverslips (22x22 mm2) coated with 0.006% type I collagen. RESULTS: These IPCs formed clusters similar to islets of Langerhans. We confirmed these clusters were positive for insulin and C-peptide by immunohistochemistry. CONCLUSION: Our data demonstrated that in vitro hUCB-derived cells generated IPCs, which can be a potential source for the treatment of diabetes via a stem cell therapy approach.

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