PDX1 Combined With NKX6.1 Induce The Bone Marrow Mesenchymal Stem Cells From Foetus To Differentiate Into Functional β-like Cells

ObjectiveTo explore a method to make the fetal bone marrow mesenchymal stem cells differentiate into the functionalβ-like cells induced by both transcription factors and cytokines,to provide a possibility of clinical transplantation in the future.Method(1)Total RNA of fetal pancreatic tissue(3-6 months old embryos from volunteers of natural miscarriage with permission) was respectively extracted.Human genes of transcription factors including Pdx1,Pax4,NeuroD1,Nkx6.1 were amplified by RT-PCR in vitro,and respectively inserted into vectors of pEGFP-N1 or pIRES to construct the recombinant plasmids,which would be used to transfect L02 cells.The expression and localization of the target genes were observed and the functional genes were selected.(2) Application of enzyme and enzyme-linked method constructed the recombined adenovirus vectors expressing both PDXI and NKX6.1 genes,which had cooperation effect and could transfect mesenchymal stem cells with high efficiency.(3) Human fetal bone marrow was douched by hyperbaric normal saline and density gradient,and centrifuged to collect mononuclear cells.Fetal bone mesenchymal stem cells (FBMSCs) were isolated and purified by differential adherent ability.The morphological figure of cells were observed and cellular growth curves were drawn.The doubling time of cells in logarithmic growth phase and cell surface markers were detected by Flow cytometry and indirect fluorescent staining.The function of osteogenesis and adipogenesis of FBMSCs was detected by cell biochemistry.(4) Recombined adenovirus vectors inserted with PDX1 and NKX6.1 (Adxsi-CMV-Pdx1/CMV-Nkx6.1) transfected FBMSCs for inductive differentiation.Cellular differentiation(phases) was identified by changes in several key genes expression profile. Various cytokines were used by turns to stimulate objective cells to further differentiate into functionalβ-like cells.(5) Streptozotocin was used to make diabetic mouse model.Differentiated functionalβ-like cells were transplant under the renal capsule of the diabetic mouse.Regulating capacity of transplanted functionalβ-like cells on mice's serum glucose were observed.Results (1) Human genes of transcription factors including Pdx1,Pax4,NeuroD1 and Nkx6.1 were amplified by RT-PCR.Recombinant plasmid vectors pEGFP-N1/Nkx6.1,pEGFP-N1/Pax4,pIRES-Pdx1/NeuroD1 were constructed and identified by restriction enzyme digestion and sequencing.Recombined vectors inserted with target genes were transfected L02 cells and the expression of target genes in cells were identified by RT-PCR, immunochistochemistry and indirect fluorescent staining.The expression of target genes mainly exited in cell nucleus.(3) The recombined adenovirus expressing both PDX1 and NKX6.1 genes (Adxsi-CMV-Pdx1/CmV-Nkx6.1) were successfully constructed and identified by restriction enzyme digestion and sequencing.These vectors could infect mesenchymai stem cells with high efficiency.(4) Primary cultured FBMSCs was passed generation by differential adherent ability. The third generation FBMSCs presented uniform long fusiform and arranged in lines.Flow cytometry detection demonstrated that the positive cellular surface markers CD₁₀₆,CD₄₄ and CD₂₉ were exceed 98%while other markers HLA-DR,CD₁₆,CD₃₄,CD₄₅ and CD₁₉ were lower than 2%.All the fetal bone mesenchymal stem cells which were not induced did not express transcription factors such as PDX1,NKX6.1,NGN3,GLUT2.(5)After induction by recombined adenovirus vectors Adxsi-CmV-Pdxl/CiV-Nkx6.1 as well as various cytokines by turns,FBMSCs were aggregated and presented islets-like bodies,nithizone staining of these cells was positive.RT-PCR showed the target genes such as Pdxl and Nkx6.1 were consistently and steadily expressed in these cells.During the second inductive phase,insulin gene began to express.During the third inductive phase,MafA and GLUT2 genes began to express,and the concentration of secreted insulin were approximately(340.4±59.3)mU/L;while under high glucose level,the concentration of secreted insulin reached to(639.8±85.1)mU/L.Immunochistochemistry and indirect fluorescent staining demonstrated that transcription factors NGN3 and mafA,which were the inductive product of Pdx1,Nkx6.1,were all expressed in target cells nucleus,while insulin,C-peptide,GLUT2 mainly located in Cytoplasm.Western blotting also proved the consistent expression of PDX1,NKX6.1,and insulin began to express in the second inductive phase.(6) Three days after transplantation of the differentiated functionalβ-like cells,serum glucose of diabetic rat can restore to normal and kept for 27 days at least.HE staining showed that the transplanted cells were under renal capsule.Cells generally appeared circular,large,rich with basophilic cytoplasid,and had plenty of secretory vesicles-like bodies pushing nucleus to one side,which showing that cells were in active secretion. Immunochistochemistry demonstrated that insulin staining were positive in cytoplasm. Indirect fluorescent staining demonstrated that insulin and C-peptide staining were positive in cytoplasm.In contrast,insulin and C-peptide could not be detected in two control groups.Conclusion(1) Plasmid vectors pEGFP-N1/Nkx6.1,pEGFP-N1/Pax4 and pIRES-Pdx1/NeuroD1 were successfully constructed.Cell transfection proved that the target genes were located in cell nucleus and had biological function.Recombined adenovirus vectors Adxsi-CMV-Pdx1/CMV-Nkx6.1 expressing both PDX1 and NKX6.1 genes that had cooperation effect were successfully constructed and selected.(2) Based on domestic and foreign methods to isolate and culture bone mesenchymal stem cells.Fetal bone marrow was douched by hyperbaric normal saline and density gradient centrifuged to collect bone mesenchymal stem cells.Then these cells were further isolated and purified by differential adherent ability.After several passages,cellular surface markers and functions identified that relatively pure fetal bone mesenchymal stem cells were collected.(3) Based on the effects of PDX1 and NKX6.1,combined with various cytokines, FBMSCs were induced to differentiate into functionalβ-like cells.The differentiated cells could actively uptake high concentration of Zn²⁺,express characteristic moleculars of functionalβ-like cells such as NGN3,MafA,GLUT2 by turns,and synthesis and secret high level of insulin.Secretion of insulin by these cells could be stimulated by high concentration of glucose.Besides,in diabetic mice model,these cells could decrease serum glucose,and kept it to a normal level,as well as promote the living condition of diabetic rat induced by streptozotocin.All the results showed that this is a successful inductive proposal to induce bone mesenchymal stem cells to differentiate to functionalβ-like cells.