Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection |
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Authors: | Phuc Van Pham Phuoc Thi-My Nguyen Anh Thai-Quynh Nguyen Vuong Minh Pham Anh Nguyen-Tu Bui Loan Thi-Tung Dang Khue Gia Nguyen Ngoc Kim Phan |
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Institution: | Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University, Ho Chi Minh City, Viet Nam |
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Abstract: | Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects. Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation. Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity. Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection. MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality. MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group). This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group). We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.3±2.5% IPCs in the combinatorial group, 3.21±2.11% in the inducer group and 0% in the control. Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration. These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment. |
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Keywords: | DMEM Dulbecco&rsquo s modified eagle medium GFP green fluorescent protein MNC mononuclear cell mRNA messenger RNA MSC mesenchymal stem cell IMDM Iscove&rsquo s modified Dulbecco&rsquo s media IPC insulin producing cell PBS phosphate buffered saline |
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