Derivation of islet‐like cells from mesenchymal stem cells using PDX1‐transducing lentiviruses

Abstract

Pancreatic duodenum homeobox protein-1 (PDX1) is a master regulatory gene in pancreatic development. Reprogramming of mesenchymal stem cells (MSCs) is a promising tool for producing insulin-producing cells. In this study, lentivirus harboring PDX1 (LV-PDX1) has been used for persistence gene expression in MSCs. The objective of this study was to evaluate the potential of lentivirus to introduce the PDX1 gene into MSCs to produce insulin-secreting cells and apply it for treatment of hyperglycemia in diabetic rats. MSCs were isolated from rat bone marrow, characterized, and transduced by LV-PDX1. Significant expressions of PDX1, neurogenin3, glucagon, glucose transporter2 (Glut2), and insulin were detected by quantitative reverse transcription-polymerase chain reaction (P < 0.05). PDX1 and insulin were detected at the protein level by immunofluorescence analysis. PDX1 could trigger a gene expression cascade that involved pancreatic endocrine differentiation and also revealed the glucose sensing ability by expressing Glut2 in high-glucose medium. The insulin secretion of MSCs(PDX1+) in the high-glucose medium was 1.75-fold higher than that secreted in the low-glucose medium (P < 0.05). MSCs(PDX1+) implanted into diabetic rats could decrease the blood glucose level from 485 mg/dL to the normal level in 3 days. This study showed MSCs(PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes.

miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells

“…According to immune regulatory properties, the cells are also used for overcoming graft-versus-host disease (GVHD) in allogeneic transplantation [31,32]. In the literature, there are many approaches that have been used to induce MSC differentiation into IPCs: various growth factors [33]; chemical substances [34,35] and transfection with viral vectors [36]. Kadam et al [34] studied the differentiation of human placenta-derived mesenchymal stem cells (hPDMSCs) into insulin-producing cells.…”

“…In the final stage, it contained α-MEM, BSA, ITS, taurine, glucagonlike peptide (GLP)-1 and nicotinamide. Talebi et al [36] studied the differentiation of MSCs from the bone marrow of Sprague-Dawley rats toward islet-like cells using PDX1-transducing lentiviruses. The cells were transplanted to diabetic rats and a reduction in blood glucose level from high to normal was revealed.…”

“…However, many problems remain in the differentiation pathway. The differentiation efficiency was low and the differentiated cells were not functional for a long time [36]. Therefore, the main goal of our study was to produce functional IPCs in a short time using a new approach.…”

Insulin producing cells established using non-integrated lentiviral vector harboring PDX1 gene

“…The cells were centrifuged at 1500 rpm for 6 min and then were resuspended in PBS at the concentration of 1 × 10 6 /mL. The fluorescent labeled antibodies (10 μL for each sample) were added and incubated for 30 min at room temperature [35,36] . The labeled cells were analyzed on a FACS Caliber (Becton-Dickinson, Franklin Lakes, NJ, United States) following labeled antibodies against human CD45-FITC/CD34-PE, CD31-FITC/CD73-PE, CD90-FITC/CD105-PE, CD11b-PE, CD44PE/ CD106-PEcy5, CD16-FITC/CD29-PE, CD14-PE/CD55-PE5 (Serotec, United States).…”

“…One day after medium change, active lentiviral vectors were released in culture medium. Culture medium was removed and viral vectors become concentrated with MILLIPORE falcons and kept in -80 ℃ for further usage [36] .…”