As an essential transcriptional activator,PDX1 plays a crucial role in pancreatic development andβ-cell function.Mutations in the PDX1 gene may lead to type 4 maturityonset diabetes of the young(MODY4)and neonatal di...As an essential transcriptional activator,PDX1 plays a crucial role in pancreatic development andβ-cell function.Mutations in the PDX1 gene may lead to type 4 maturityonset diabetes of the young(MODY4)and neonatal diabetes mellitus.However,the precise mechanisms underlying MODY4 remain elusive due to the paucity of clinical samples and pronounced differences in pancreatic architecture and genomic composition between humans and existing animal models.In this study,three PDX1-mutant cynomolgus macaques were generated using CRISPR/Cas9 technology,all of which succumbed shortly postpartum,exhibiting pancreatic agenesis.Notably,one tri-allelic PDX1-mutant cynomolgus macaque(designated as M4)developed a pancreas,whereas the two monoallelic PDX1-mutant cynomolgus macaques displayed no anatomical evidence of pancreatic formation.RNA sequencing of the M4 pancreas revealed substantial molecular changes in both endocrine and exocrine functions,indicating developmental delay and PDX1haploinsufficiency.A marked change in m6A methylation was identified in the M4 pancreas,confirmed through cultured PDX1-mutantisletorganoids.Notably,overexpression of the m6A modulator METTL3 restored function in heterozygous PDX1-mutant islet organoids.This study highlights a novel role of m6A methylation modification in the progression of MODY4 and provides valuable molecular insights for preclinical research.展开更多
胰腺-十二指肠同源盒1基因(pancreatic and duodenal homeobox 1,PDX1)是决定胰腺分化和发育,调控胰岛素的关键基因。PDX1的缺失及下调可诱发糖尿病的发生和发展,而对其上调则能够调节胰腺发育、促β细胞分化和胰岛素分泌,发挥治疗糖尿...胰腺-十二指肠同源盒1基因(pancreatic and duodenal homeobox 1,PDX1)是决定胰腺分化和发育,调控胰岛素的关键基因。PDX1的缺失及下调可诱发糖尿病的发生和发展,而对其上调则能够调节胰腺发育、促β细胞分化和胰岛素分泌,发挥治疗糖尿病作用。PDX1已然成为糖尿病治疗药物开发的新靶点。越来越多的天然产物被发现具有上调PDX1的作用且表现出抗糖尿病活性。然而,目前尚未有相关文献进行总结报道。本文通过检索CNKI、Pubmed等数据库,对调控PDX1的天然产物抗糖尿病作用的相关研究进行归纳和总结,以期为靶向调控PDX1的新型抗糖尿病药物开发提供思路和参考。展开更多
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
Pdx1(pancreatic and duodenal homeobox 1)是一种同源框转录因子,在胰腺发育及β细胞功能维持中起重要调控作用。研究发现,Pdx1基因纯合缺失会导致胰腺发育不全;在β细胞成熟与分化过程中,Pdx1通过协同其他因子或以顺式调控的方式参与...Pdx1(pancreatic and duodenal homeobox 1)是一种同源框转录因子,在胰腺发育及β细胞功能维持中起重要调控作用。研究发现,Pdx1基因纯合缺失会导致胰腺发育不全;在β细胞成熟与分化过程中,Pdx1通过协同其他因子或以顺式调控的方式参与调节;此外,Pdx1可以直接与胰岛素启动子结合,调节胰岛素合成与分泌。近来针对Pdx1的研究表明,它在β细胞重塑、基因编辑及药物开发等方面具有重要作用。现就Pdx1在胰腺发育中的作用机制及其在糖尿病治疗中的进展进行综述。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
基金supported by the National Key R&D Program of China(2018YFA0801404,2023YFC3403400)National Natural Science Foundation of China(81941006,32371190,32370878)+2 种基金Guangdong Special Support Program(2019BT02Y276,2024A1515012868)Double First-Class Discipline Promotion Project(2023B10564003)Program for Scientific Research Start-up Funds of Guangdong Ocean University(060302052408)。
文摘As an essential transcriptional activator,PDX1 plays a crucial role in pancreatic development andβ-cell function.Mutations in the PDX1 gene may lead to type 4 maturityonset diabetes of the young(MODY4)and neonatal diabetes mellitus.However,the precise mechanisms underlying MODY4 remain elusive due to the paucity of clinical samples and pronounced differences in pancreatic architecture and genomic composition between humans and existing animal models.In this study,three PDX1-mutant cynomolgus macaques were generated using CRISPR/Cas9 technology,all of which succumbed shortly postpartum,exhibiting pancreatic agenesis.Notably,one tri-allelic PDX1-mutant cynomolgus macaque(designated as M4)developed a pancreas,whereas the two monoallelic PDX1-mutant cynomolgus macaques displayed no anatomical evidence of pancreatic formation.RNA sequencing of the M4 pancreas revealed substantial molecular changes in both endocrine and exocrine functions,indicating developmental delay and PDX1haploinsufficiency.A marked change in m6A methylation was identified in the M4 pancreas,confirmed through cultured PDX1-mutantisletorganoids.Notably,overexpression of the m6A modulator METTL3 restored function in heterozygous PDX1-mutant islet organoids.This study highlights a novel role of m6A methylation modification in the progression of MODY4 and provides valuable molecular insights for preclinical research.
文摘胰腺-十二指肠同源盒1基因(pancreatic and duodenal homeobox 1,PDX1)是决定胰腺分化和发育,调控胰岛素的关键基因。PDX1的缺失及下调可诱发糖尿病的发生和发展,而对其上调则能够调节胰腺发育、促β细胞分化和胰岛素分泌,发挥治疗糖尿病作用。PDX1已然成为糖尿病治疗药物开发的新靶点。越来越多的天然产物被发现具有上调PDX1的作用且表现出抗糖尿病活性。然而,目前尚未有相关文献进行总结报道。本文通过检索CNKI、Pubmed等数据库,对调控PDX1的天然产物抗糖尿病作用的相关研究进行归纳和总结,以期为靶向调控PDX1的新型抗糖尿病药物开发提供思路和参考。
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.
文摘Pdx1(pancreatic and duodenal homeobox 1)是一种同源框转录因子,在胰腺发育及β细胞功能维持中起重要调控作用。研究发现,Pdx1基因纯合缺失会导致胰腺发育不全;在β细胞成熟与分化过程中,Pdx1通过协同其他因子或以顺式调控的方式参与调节;此外,Pdx1可以直接与胰岛素启动子结合,调节胰岛素合成与分泌。近来针对Pdx1的研究表明,它在β细胞重塑、基因编辑及药物开发等方面具有重要作用。现就Pdx1在胰腺发育中的作用机制及其在糖尿病治疗中的进展进行综述。
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.
