Differentiation Of Bone Marrow Mesenchymal Stem Cells And IPSCs Into Insulin-producing Cells Via Adenoviral Transfection Of PDX-1, NeuroD1and MafA

Objective To evaluate the effect of insulin gene transcription regulators PDX-1,NeuroD1and MafA on the differentiation of bone marrow mesenchymal stem cells（mBMSCs）and induced pluripotent stem cells (iPSCs) into insulin-producing cells.Andto confer the new approach of cell transplantation therapy for type I diabetes.Methods⑴Coding regional sequences of transcription factors PDX-1, NeuroD1and MafAwere obtained by total gene synthesis. Then made a confirm by sequencing. Splicingsegments of target genes were augmented and jointed together with the internal ribosomeentry site sequence-green fluorescent protein（IRES-GFP） respectively,and to obtain thegene fragments expressing target genes and GFP. The next the plasmids harboring targetgenes were restructuring to adenovirus vectors. The recombinant adenovirus vectors whichharbored target genes were transfected into packaging cell line293A. And the adenoviruswhich had aggression was harvested. The titer of the adenovirus was determined byimmune method.(2)Mouse Embryonic Fibroblasts (MEFs) were infected with lentivirus(LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection. Toconfirm iPSCs pluripotency, Morphology of iPSCs, mES cell-specific cell surface markers,including Nanog, SSEA-1and Rex-1, differentiation experiments both in vitro and in vivowere detected.⑶mBMSCs and iPSCs were infected with adenovirus (Ad-mPDX-1-IRES-GFP, Ad–mNeuroD1-IRES-GFP and Ad-mMafA-IRES-GFP), and then differentiated intoinsulin-producing cells in vitro. RT-PCR was applied to detecting target genes and insulingene expression, immunofluorescence to identifying the presence and location of insulinprotein, and mouse insulin enzyme-linked immunosorbent assay (ELISA) for evaluatingthe secretory volume of insulin at different concentration of glucose.⑷Diabetic mice were transplanted with infection mBMSCs and iPSCs under the liverparenchyma. The grafts were analyzed by Fasting plasma glucose were used to assess thefunctions exertion of engrafted cell in vivo and revealed the therapeutic effect.Results⑴Recombinant adenovirus vectors expressing PDX-1,NeuroD1and MafA werelinearized with Pac I, separated by agarose gel electrophoresis.2,000bp of small strap andnearly35,000bp a of big strap were appeared, with the expected results are consistent. Itproved the exactitude of homologous adenovirus vectors.⑵MEFs were infected with the four reprogramming factors(LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection. TheiPSCs derived from MEFs were able to grow into clones with clear borders and wereconfirmed the expression of mES cell-specific cell surface markers, including Nanog,SSEA-1and Rex-1. Differentiation experiments showed that suspension cultures of allclones formed EBs in vitro and all three layers in vivo.⑶The β-cell-specific transcriptional regulators and the insulin gene were expressed inmBMSCs after infection. Immunofluorescence analyses documented the activation ofexpression of insulin in the cytoplasm of differentiated cells. The release of a significantinsulin content by these cells was detected in response to a certain concentrations ofglucose stimulation.⑷Immunohistochemistry was performed to detect the expression of insulin in theliver tissue of diabetic mice and it exhibited positive staining of insulin. The results offasting plasma glucose demonstrated the ability of these insulin-producing cells-transplanted mice to dispose of a glucose load.Conclusions⑴MEFs which were infected with the four reprogramming factors(LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) can be reprogrammed into iPSCs.⑵The combination of PDX-1, NeuroD1and MafA markedly induces insulinbiosynthesis and secretion in mBMSCs and iPSCs.⑶Transplantation of mBMSCs and iPSCs modified by transcriptional factors hastherapeutical effect on type I diabetes and thereby is a novel approach efficiently induceinsulin-producing surrogateβ cells.