Studies On Insulin-producing Cells Induced From Mouse Adipose Tissue-derived Mesenchymal Stem Cells

Islet transplantation, including b-cells, has proven to be effective fordiabetes in many recent studies; however, this treatment strategy requiressufficient organ donors. One attractive approach for the generation ofb-cells is to utilize the expansion and differentiation of cells frompancreatic stem cells (PSCs), which are closely associated to the β-cellslineage. In this study, mADSCs were chosen as target cells fortransduction, because of their ability to differentiate into multiple celltypes[12,13], and their ability to elude detection by the host’s immunesystem[14].In our study, Lentiviral overexpression vectors harboring Pdx1(pancreatic and duodenal homeobox-1),MafA(V-maf musculoaponeuroticfibrosarcoma oncogene homolog A) and NeuroD1(neural differentiationfactor-1) were constructed, respectively. Then we packed lentivirus ofPdx1, MafA and NeuroD1using293T cells and concentrated. mADSCswere induced with different combinations of Pdx1, MafA and NeuroD1genes. At15d after transfection, IPCs induced by different ways wereidentified with DTZ staining. The concentration of glucose were detectedduring0-120min after stimulation. Expressions of Pdx1, MafA,NeuroD1, Ngn3and Insulin2as specific marker genes were investigatedat mRNA level.We reported that1. Lentiviral overexpression vectors, pHBLV-CMV-IRES-ZsGreen-Pdx1、pHBLV-CMV-PGK-RFP-MafA and pHBLV- CMV-PGK-RFP-NeuroD1, in which their purpose fragment gene sequencescontained exactly the same as that of coding sequence of all the genes inmice, indicating that three genes lentiviral overexpression vectors weresuccessful constructed;2. At15d after transduction, IPCs clones showedpositive staining of DTZ and expressed insulin biosynthesis and secretionrelated genes;3. Stimulate IPCs with high glucose DMEM mediusm,IPCs induced with a combination of3factors showed the most effectiveregulation and the lowest glucose concentration compared with othergroups.In our study, a combination of three transcription factors, which allplay a crucial role in glucose induction of insulin gene transcription andpancreatic β-cell function, were delivered into mADSCs on lentiviralvectors. We investigated whether important transcription factors (Pdx-1,MafA and NeuroD1) in islet cells could be efficiently transduced intomADSCs using lentiviral vectors and found that the transduced cells weredifferentiated into insulin-producing cells. These data suggest that thetransduction of the combination of PDX-1, NeuroD, and MafA showedthe most efficiency out of all the different combinations and showed themost effective regulation of glucose concentration.