The Experiment Of Polygene-modified Induced Pluripotent Stem Cells Differentiated Into Insulin-producing Cells

Objective To evaluate the effect of insulin gene transcription regulators PDX-1, NeuroD and MafA on the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells. And to confer the new approach of cell transplantation therapy for type1diabetes.Methods(1) Mouse Embryonic Fibroblasts (MEFs) were infected with lentivirus (LV-efla-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection. To confirm iPSCs pluripotency, Morphology of iPSCs, mES cell-specific cell surface markers, including Nanog, SSEA-1and Rex-1, differentiation experiments both in vitro and in vivo were detected.(2) iPSCs were infected with adenovirus (Ad-mPDX-1-IRES-GFP, Ad-mNeuroD-IRES-GFP and Ad-mMafA-IRES-GFP), and then differentiated into insulin-producing cells in vitro. RT-PCR was applied to detecting target genes and insulin gene expression, immunofluorescence to identifying the presence and location of insulin protein, and mouse insulin enzyme-linked immunosorbent assay (ELISA) for evaluating the secretory volume of insulin at different concentration of glucose. (3) Diabetic mice were transplanted with infection iPSCs under the liver parenchyma. The grafts were analyzed by immunohistochemistry for the presence of insulin-producing cells. Fasting plasma glucose were used to assess the functions exertion of engrafted cell in vivo and revealed the therapeutic effect.Results(1) MEFs were infected with the four reprogramming factors (LV-efla-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection. The iPSCs derived from MEFs were able to grow into clones with clear borders and were confirmed the expression of mES cell-specific cell surface markers, including Nanog, SSEA-1and Rex-1. Differentiation experiments showed that suspension cultures of all clones formed EBs in vitro and all three layers in vivo.(2) iPSCs were infected with adenovirus (Ad-mPDX-1-IRES-GFP, Ad-mNeuroD-IRES-GFP and Ad-mMafA-IRES-GFP) at a multiplicity of infection. The β cell-specific genes were detected in polygene-modified induced pluripotent stem cells after infection. RT-PCR results showed that polygene-modified iPSCs and pancreatic β cell line MIN6have similar gene expression. Immunofluorescence analyses confirmed the activation of expression of insulin in the cytoplasm of differentiated cells. Mouse insulin enzyme-linked immunosorbent assay (ELISA) showed polygene-modified iPSCs have a Satisfactory response to different concentrations of glucose.(3) Immunohistochemistry was performed to detect the expression of insulin in the liver tissue of diabetic mice and it exhibited positive staining of insulin. The results of fasting plasma glucose demonstrated the ability of these insulin-producing cells-transplanted mice to dispose of a glucose load.Conclusions (1)、MEFs which were infected with the four reprogramming factors (LV-efla-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) can be reprogrammed into iPSCs.(2)、The combination of PDX-1, NeuroD and MafA markedly induces insulin biosynthesis and secretion in iPSCs.(3)、Transplantation of polygene-modified iPSCs have therapeutical effect on type1diabetes and thereby is a novel approach efficiently induce insulin-producing surrogate β cells.