AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem c...AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.展开更多
AIM:To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm thatβ-cell progenitors reside within the pancreatic epithelium.METHODS:Due to mesenchymal stem cell(MSC)expansion and overgrowth,prog...AIM:To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm thatβ-cell progenitors reside within the pancreatic epithelium.METHODS:Due to mesenchymal stem cell(MSC)expansion and overgrowth,progenitor cells within the pancreatic epithelium cannot be characterized in vitro,thoughβ-cell dedifferentiation and expansion of MSC intermediates via epithelial-mesenchymal transition(EMT)may generateβ-cell progenitors.Pancreatic epithelial cells from endocrine and non-endocrine tissue were expanded and differentiated in a novel pancreatic epithelial expansion medium supplemented with growth factors known to support epithelial cell growth(dexamethasone,epidermal growth factor,3,5,3’-triiodo-l-thyronine,bovine brain extract).Cells were also infected with a single and dual lentiviral reporter prior to cell differentiation.Enhanced green fluorescent protein was controlled by the rat Insulin 1 promoter and the monomeric red fluorescent protein was controlled by the mouse PDX1 promoter.In combination with lentiviral tracing,cells expanded and differentiated in the pancreatic medium were characterized by flow cytometry(BD fluorescence activated cell sorting),immunostaining and real-time polymerase chain reaction(PCR)(7900HT Fast Realtime PCR System).RESULTS:In the presence of 10%serum MSCs rapidly expand in vitro while the epithelial cell population declines.The percentage of vimentin+cells increased from 22%±5.83%to 80.43%±3.24%(14 d)and99.00%±0.0%(21 d),and the percentage of epithelial cells decreased from 74.71%±8.34%to 26.57%±9.75%(14 d)and 4.00%±1.53%(21 d),P<0.01 for all time points.Our novel pancreatic epithelial expansion medium preserved the epithelial cell phenotype and minimized epithelial cell dedifferentiation and EMT.Cells expanded in our epithelial medium contained significantly less mesenchymal cells(vimentin+)compared to controls(44.87%±4.93%vs 95.67%±1.36%;P<0.01).During cell differentiation lentiviral reporting demonstrated that,PDX1+and insulin+cells were localized within adherent epithelial cell aggregates compared to controls.Compared to starting islets differentiated cells had at least two fold higher gene expression of PDX1,insulin,PAX4 and RFX(P<0.05).CONCLUSION:PDX1+cells were confined to adherent epithelial cell aggregates and not vimentin+cells(mesenchymal),suggesting that EMT is not a mechanism for generating pancreatic progenitor cells.展开更多
目的研究高糖毒性对INS-1细胞胰岛素基因及其转录因子PDX-1和Maf A表达的影响,及NF-κB抑制剂吡咯烷二硫基甲酸盐(PDTC)的保护作用,探讨"糖毒性"的生化机制。方法分别以4.0 mmol/L葡萄糖(NG组)、16.7mmol/L葡萄糖(HG组)及16.7...目的研究高糖毒性对INS-1细胞胰岛素基因及其转录因子PDX-1和Maf A表达的影响,及NF-κB抑制剂吡咯烷二硫基甲酸盐(PDTC)的保护作用,探讨"糖毒性"的生化机制。方法分别以4.0 mmol/L葡萄糖(NG组)、16.7mmol/L葡萄糖(HG组)及16.7 mmol/L葡萄糖+50μmol/L PDTC(HG+P组)培养INS-1细胞,48 h后,离心收集细胞,提取总RNA,以real-ti me PCR法检测胰岛素基因及转录因子PDX-1和Maf A的mRNA水平。结果HG组与NG组比较,胰岛素mRNA的表达下降了64.9%(P<0.05);PDX-1 mRNA的表达下降了82.3%(P<0.01);Maf A mRNA的表达下降了80.9%(P<0.01)。HG+P组与HG组相比,胰岛素mRNA的表达则提高了6.67倍(P<0.05);PDX-1mRNA的表达提高了7.52倍(P<0.05);Maf A mRNA的表达提高了5.70倍(P<0.01);而与NG组相比,胰岛素、PDX-1和Maf A mRNA的表达差异均无统计学意义(均P>0.05)。结论"糖毒性"可以通过激活INS-1细胞内的NF-κB途径,使胰岛素转录因子PDX-1和Maf A的表达下降,而导致胰岛素基因表达下降,抑制NF-κB可起到保护作用。展开更多
Prostaglandin E-2(PGE(2)) is a well-known mediator of beta-cell dysfunction in both type 1 and type 2 diabetes.We recently reported that down-regulation of the Akt pathway activity is implicated in PGE(2)-induced panc...Prostaglandin E-2(PGE(2)) is a well-known mediator of beta-cell dysfunction in both type 1 and type 2 diabetes.We recently reported that down-regulation of the Akt pathway activity is implicated in PGE(2)-induced pancreatic beta-cell dysfunction.The aim of this study was to further dissect the signaling pathway of this process in pancreatic beta-cell line HIT-T15 cells and primary mouse islets.We found that PGE(2) time-dependently increased the c-Jun N-terminal kinase(JNK) pathway activity.JNK inhibition by the JNK-specific inhibitor SP600125 reversed PGE(2)-inhibited glucose-stimulated insulin secretion(GSIS).PGE(2) induced dephosphorylation of Akt and FOXO1, leading to nuclear localization and transactivation of FOXO1.Activation of FOXO1 induced nuclear exclusion but had no obvious effect on the whole-cell protein level of pancreatic and duodenal homeobox 1(PDX1).However, these effects were all attenuated by JNK inhibition.Furthermore, adenovirus-mediated overexpression of dominant-negative(DN)FOXO1 abolished whereas constitutively active(CA)-FOXO1 mimicked the effects of PGE(2) on GSIS in isolated mouse islets.In addition, we demonstrated that DN-JNK1 but not DN-JNK2 or CA-Akt abolished the PGE(2)-induced AP-1 luciferase reporter activity, whereas DN-JNK1 and CA-Akt but not DN-JNK2 reversed the effect of PGE(2) on FOXO1 transcriptional activity, and overexpression of DN-JNK1 rescued PGE(2)-impaired GSIS in mouse islets.Our results revealed that activation of the JNK is involved in PGE(2)induced beta-cell dysfunction.PGE(2)-mediated JNK1 activation, through dephosphorylation of Akt and FOXO1, leads to nuclear accumulation of FOXO1 and nucleocytoplasmic shuttling of PDX1, finally resulting in defective GSIS in pancreatic beta-cells.展开更多
基金Supported by National Institute of Genetic Engineering and Biotechnology,Ministry of Science Research and Technology,Tehran,Iran
文摘AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.
基金Supported by Canadian Institutes of Health Research,No.MOP8030the Alberta Diabetes Institute
文摘AIM:To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm thatβ-cell progenitors reside within the pancreatic epithelium.METHODS:Due to mesenchymal stem cell(MSC)expansion and overgrowth,progenitor cells within the pancreatic epithelium cannot be characterized in vitro,thoughβ-cell dedifferentiation and expansion of MSC intermediates via epithelial-mesenchymal transition(EMT)may generateβ-cell progenitors.Pancreatic epithelial cells from endocrine and non-endocrine tissue were expanded and differentiated in a novel pancreatic epithelial expansion medium supplemented with growth factors known to support epithelial cell growth(dexamethasone,epidermal growth factor,3,5,3’-triiodo-l-thyronine,bovine brain extract).Cells were also infected with a single and dual lentiviral reporter prior to cell differentiation.Enhanced green fluorescent protein was controlled by the rat Insulin 1 promoter and the monomeric red fluorescent protein was controlled by the mouse PDX1 promoter.In combination with lentiviral tracing,cells expanded and differentiated in the pancreatic medium were characterized by flow cytometry(BD fluorescence activated cell sorting),immunostaining and real-time polymerase chain reaction(PCR)(7900HT Fast Realtime PCR System).RESULTS:In the presence of 10%serum MSCs rapidly expand in vitro while the epithelial cell population declines.The percentage of vimentin+cells increased from 22%±5.83%to 80.43%±3.24%(14 d)and99.00%±0.0%(21 d),and the percentage of epithelial cells decreased from 74.71%±8.34%to 26.57%±9.75%(14 d)and 4.00%±1.53%(21 d),P<0.01 for all time points.Our novel pancreatic epithelial expansion medium preserved the epithelial cell phenotype and minimized epithelial cell dedifferentiation and EMT.Cells expanded in our epithelial medium contained significantly less mesenchymal cells(vimentin+)compared to controls(44.87%±4.93%vs 95.67%±1.36%;P<0.01).During cell differentiation lentiviral reporting demonstrated that,PDX1+and insulin+cells were localized within adherent epithelial cell aggregates compared to controls.Compared to starting islets differentiated cells had at least two fold higher gene expression of PDX1,insulin,PAX4 and RFX(P<0.05).CONCLUSION:PDX1+cells were confined to adherent epithelial cell aggregates and not vimentin+cells(mesenchymal),suggesting that EMT is not a mechanism for generating pancreatic progenitor cells.
文摘目的研究高糖毒性对INS-1细胞胰岛素基因及其转录因子PDX-1和Maf A表达的影响,及NF-κB抑制剂吡咯烷二硫基甲酸盐(PDTC)的保护作用,探讨"糖毒性"的生化机制。方法分别以4.0 mmol/L葡萄糖(NG组)、16.7mmol/L葡萄糖(HG组)及16.7 mmol/L葡萄糖+50μmol/L PDTC(HG+P组)培养INS-1细胞,48 h后,离心收集细胞,提取总RNA,以real-ti me PCR法检测胰岛素基因及转录因子PDX-1和Maf A的mRNA水平。结果HG组与NG组比较,胰岛素mRNA的表达下降了64.9%(P<0.05);PDX-1 mRNA的表达下降了82.3%(P<0.01);Maf A mRNA的表达下降了80.9%(P<0.01)。HG+P组与HG组相比,胰岛素mRNA的表达则提高了6.67倍(P<0.05);PDX-1mRNA的表达提高了7.52倍(P<0.05);Maf A mRNA的表达提高了5.70倍(P<0.01);而与NG组相比,胰岛素、PDX-1和Maf A mRNA的表达差异均无统计学意义(均P>0.05)。结论"糖毒性"可以通过激活INS-1细胞内的NF-κB途径,使胰岛素转录因子PDX-1和Maf A的表达下降,而导致胰岛素基因表达下降,抑制NF-κB可起到保护作用。
文摘Prostaglandin E-2(PGE(2)) is a well-known mediator of beta-cell dysfunction in both type 1 and type 2 diabetes.We recently reported that down-regulation of the Akt pathway activity is implicated in PGE(2)-induced pancreatic beta-cell dysfunction.The aim of this study was to further dissect the signaling pathway of this process in pancreatic beta-cell line HIT-T15 cells and primary mouse islets.We found that PGE(2) time-dependently increased the c-Jun N-terminal kinase(JNK) pathway activity.JNK inhibition by the JNK-specific inhibitor SP600125 reversed PGE(2)-inhibited glucose-stimulated insulin secretion(GSIS).PGE(2) induced dephosphorylation of Akt and FOXO1, leading to nuclear localization and transactivation of FOXO1.Activation of FOXO1 induced nuclear exclusion but had no obvious effect on the whole-cell protein level of pancreatic and duodenal homeobox 1(PDX1).However, these effects were all attenuated by JNK inhibition.Furthermore, adenovirus-mediated overexpression of dominant-negative(DN)FOXO1 abolished whereas constitutively active(CA)-FOXO1 mimicked the effects of PGE(2) on GSIS in isolated mouse islets.In addition, we demonstrated that DN-JNK1 but not DN-JNK2 or CA-Akt abolished the PGE(2)-induced AP-1 luciferase reporter activity, whereas DN-JNK1 and CA-Akt but not DN-JNK2 reversed the effect of PGE(2) on FOXO1 transcriptional activity, and overexpression of DN-JNK1 rescued PGE(2)-impaired GSIS in mouse islets.Our results revealed that activation of the JNK is involved in PGE(2)induced beta-cell dysfunction.PGE(2)-mediated JNK1 activation, through dephosphorylation of Akt and FOXO1, leads to nuclear accumulation of FOXO1 and nucleocytoplasmic shuttling of PDX1, finally resulting in defective GSIS in pancreatic beta-cells.
