| In vitro differentiation of pancreatic duct stem cells(PDSCs)into insulin-secreting,?-like cells(BLCs)not only deliberate the molecular developmental mechanisms but can also serve as a pool for insulin-producing cells(IPCs)for cell therapy of diabetes.Islet cells are formed during the development of pancreatic ductal networks from multipotent and bipotent progenitors found in the pancreatic duct epithelium.Adult pancreatic stem cells are found in fetal pancreatic ductal networks and persist in adult pancreatic ducts as progenitors of pancreatic endocrine cells.The leading players regulating the development of mature islet cells from the pancreatic stem or progenitor cells are genes and transcription factors that govern the spatiotemporal regulation of crucial molecular processes of islet development.Many previous studies have reported multipotent stem cells or progenitors in the pancreatic ductal system of the adult pancreas.Many researchers have used pancreatic ductal stem cells for developing?-like cells in vitro using small molecules and growth factors.However,in vitro development of bonafide?cells necessitates replicating in vivo developmental signaling pathways,which is a difficult task.As a result,until recently,the in vitro developed BLCs were not pure or mature enough to be used as a clinical option for?-cell replacement.Our team has already isolated and developed a cell line of pancreatic ductal epithelial stem cells(PDESCs)from an adult rat and demonstrated their morphological,karyotyping,expression of stem cell-specific genes at m RNA and protein levels,and multilineage potential.This study shows the effect of various concentrations of islet neogenesis-associated protein-pentadecapeptide(INGAP-PP),nicotinamide(NIC),and vitamin D3(Vit D3)supplementation in basic culture media(BCM)for differentiating rat PDESCs into BLCs.First,the optimal concentrations of INGAP-PP,NIC,and Vit D3 were screened independently,and then used those optimized concentrations in various combinations to develop an efficient culture protocol.Then the cell morphology,gene expression of critical transcription factors of?cells,and glucose-stimulated insulin secretion(GSIS)results of different groups were analyzed to compare the effect of different factors alone and in combination on the differentiation of PDESCs into BLCs.This study is further divided into four parts,each of which provides a theoretical foundation for in vitro islet?-cell development;Experiment 1:INGAP induced the development of?-like cells from rat pancreatic ductal stem cells in vitroThe goal line of this study was to find the optimal concentration of INGAP-PP(ING) for differentiating adult rat PDESCs into BLCs.The 12th generation PDESCs were cultivated,expanded,and divided into control group and four INGAP supplemented inducement groups.The BCM(DMEM/F12+10%FBS+1%penicillin-streptomycin)was used as inducement media for the control group,and four ING,indument groups,were supplemented with 100n M,150n M,200n M,and 250n M of INGAP-PP in BCM.The rat PDESCs were cultivated for 28days,and morphological changes were observed every three days using microscopy.Dithizone(DTZ)staining,q RT-PCR,immunofluorescence(IF),and GSIS were used to check the identity of induced cells,the level of insulin 1(INS1)and pancreatic and duodenal homeobox 1(Pdx1)m RNA expression,intracellular protein expression of insulin,and Pdx1,and the quantity of released insulin and C-peptide,respectively,after inducement for 28 days.The results showed that the induced cells in the ING treated groups grew like islet-like cell clusters(ICCs)with DTZ staining positive cells,whereas the control group did not develop ICCs.Furthermore,the m RNA expressions of INS1 and Pdx1 in INGAP supplemented groups were suggestively higher(p<0.01),contrasting to control group,and the induced cells in the ICCs showed insulin and Pdx1 co-expression.When induced cells in the ING supplemented groups were given the challenge of low and high glucose,they secreted suggestively higher(p<0.01)insulin and C-peptide amounts than the control group.Combined results of above-mentioned assays showed that 150n M ING was efficient to induce the production of insulin-secreting?-like cells from rat PDESCs in the lab.Experiment 2:Nicotinamide induced the development of?-like cells from rat pancreatic ductal stem cells in vitroThe study's main objective was to figure out the best nicotinamide concentration for differentiating rat PDESCs into BLCs.For this experiment,12th generation PDESCs were cultured,proliferated,and divided into control group,and four NIC supplemented inducement groups.For the control group,BCM was used as inducement media,while for the four NIC inducement groups,BCM was supplemented with 5m M,10m M,15m M,and 20m M of NIC.PDESCs were cultivated at 37°C for 28 days in a 5%CO2 incubator,and morphological changes after every three days were discerned using microscopy.DTZ staining,q RT-PCR,IF,and GSIS were used to identify the induced cells,INS1 and Pdx1 expression levels,the intracellular insulin and Pdx1 protein expression,and released C-peptide and insulin amounts after inducing for 28 days,respectively.The induced cells in the NIC-treated groups grew like ICCs with DTZ staining positive cells,while the control group did not form ICCs.Furthermore,the NIC supplemented groups had significantly higher(p<0.01)INS1 and Pdx1m RNA expression than control group,and the ICCs of inducement groups had insulin and Pdx1co-expression.When induced cells in the NIC supplemented groups were subjected to low and high glucose concentrations,they secreted significantly more insulin and C-peptide(p<0.01)in contrast to the control group.These findings of the above-mentioned assays reveal that NIC was effective in inducing PDESCs'differentiation into ICCs containing BLCs and that a concentration of 10m M was found to be optimal for this purpose.Experiment 3:Vitamin D3 induced the development of?-like cells from rat pancreatic ductal stem cells in vitroThis study intended to screen the efficient concentration of Vit D3 for inducing the differentiation of adult rat PDESCs into BLCs.For this purpose,the 12th generation PDESCs were proliferated and divided into control group,and four Vit D3 supplemented inducement groups,and every group had 36 replicates.The inducement media consisted of the BCM for the control group,and four Vit D3 indument groups were supplemented with 10-5M Vit D3,10-6M Vit D3,10-7M Vit D3,and 10-8M Vit D3 in BCM.The cells were cultured continuously over28 days,and the morphological changes were observed through microscopy every three days.After the 28d inducement,DTZ staining,q RT-PCR,IF,and GSIS were executed to reveal that induced cells in Vit D3 supplemented groups were grown like ICCs containing DTZ staining positive cells while the control group lacked the formation of ICCs.Further,the m RNA expression of INS1 and Pdx1 was considerably higher(p<0.01)in Vit D3 inducement groups as against the control group.Also,the induced cells in the ICCs of Vit D3 supplemented groups were also immunoreactive to insulin and Pdx1.When exposed to low and high glucose,the induced cells in Vit D3 supplemented groups secreted much higher levels of insulin and C-peptide related to the control group.Thus,adding Vit D3 to basic culture media can lead to the development of insulin-secreting BLCs by differentiation of PDESCs,with an adjusted concentration of 10-6M or 10-7M for that purpose.Experiment 4:ING,NIC,and Vit D3 induced the development of?-like cells from rat pancreatic ductal stem cells in vitroThis study's objective was to determine which combination of ING,NIC,and Vit D3 was best for converting rat PDESCs into BLCs.12th generation PDESCs were proliferated and divided into control and I+V,N+V,N+I,and N+I+V inducement groups.For the control group BCM was used as inducement media,while for the I+V,N+V,N+I,and N+I+V inducement groups,BCM was supplemented with 150n M ING+10-7M Vit D3,10m M NIC+10-7M Vit D3,10m M NIC+150n M ING,and 10m M NIC+150n M ING+10-7M Vit D3respectively.PDESCs were cultured for 28 days at 37°C within a moistened 5%CO2 incubator,and morphological changes were observed every three days using a microscope.After 28 days of induction,DTZ staining,q RT-PCR,IF,and GSIS were used to identify the induced cells,measure the INS1 and Pdx1 expression levels,and visualize the intracellular insulin and Pdx1protein,and to quantify the released C-peptide and insulin,respectively.The induced cells in the I+V,N+V,N+I,and N+I+V groups were alike to islets on the 28th day of inducement and stained positive for DTZ staining.Insulin and Pdx1 were co-expressed in cells from the I+V,N+V,N+I,and N+I+V groups.However,upon comparison with the control group,I+V,and N+V groups,the m RNA expression levels of INS1 and Pdx1 were meaningfully higher(p<0.01)in the N+I and N+I+V groups.Additionally,the N+I group showed increased INS1and Pdx1 expression levels in contrast to the N+I+V group.When induced cells in the N+I and N+I+V groups were stimulated with low and high glucose concentrations,GSIS assay revealed that they released higher amounts of C-peptide and insulin.Thus,the combination of NIC(10m M)+ING(150n M)was discovered to be the most effective for developing in vitro BLCs from PDESCs.Overall,the purpose of this study was to investigate the effect of ING,NIC,and Vit D3 on the differentiation inducement ability of PDESCs into BLCs singly,pairly,or all together.The results of studies as the single-molecule usage deployed the efficient concentration responsible for differentiation of PDESCs into BLCs.In the next step,the pairly and altogether usage of defined efficient concentrations of ING,NIC,and Vit D3 revealed that all combinations were suitable for the differentiation of PDESCs into BLCs.Still,the best one was of NIC and ING because of the high expression of INS1 and Pdx1 in this group. |
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