Studies On Mesenchymal Stem Cell Isolation, Culture And Induced Differentiation To Insulin-Secreting Cells In Vitro

Objective: The in vitro studies on human mesenchymal stem cells (MSCs) isolation, culture and induced differentiation to insulin-secreting cells as well as the identification of the induced insulin-secreting cells can provide sufficient source of seed cells on stem cell transplantation of diabetes therapy in clinical. Methods: 1. Whole bone marrow and MSCs Percoll density gradient centrifugation isolated MSCs were cultured with the same conditions. The number of cell clone, cell morphology, cell superficial mark and the differentiation into fat cells were compared between these two MCS isolation culture methods. 2. After three to five passages, the cultured MSCs obtained from whole bone marrow culture were used to induce differentiation into islet-like cells through three developmental stages with culture medium supplemented with thioglycol, glutamine, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and nicotinamide. 3. Morphological changes of MSCs undergoing differentiation were analyzed under phase contrast microscopy. Pancreatic and Duodenal Homeobox 1 (PDX-1) expression was detected by indirect immunofluoresence assay. Isletβ-like cell clusters were identified by positive dithizone staining. The insulin secretions were measured by electrochemiluminescence immunoassay (ECLIA). Results: 1. The number of adhesive cell clone by whole bone marrow culture is more than by Percoll density gradient centrifugation (P < 0.05). There is no remarkable differentiation of morphological expression, positive CD44 expression and negative CD34 expression about MSCs using the two methods (P>0.05). They are both can be differentiated into fat cells by insulin and dexamethasone. 2. MSCs induced with thioglycol and nicotinamide gradually became round and formed clusters. Cells of the 2nd stage expressed the PDX-1 gene product and at the 3rd stage formed islet-like cell clusters that exhibited positive dithizone staining and secreted insulin under glucose treatment. In contrast, uninduced MSCs were long adhesive shuttles and did not exhibit PDX-1 expression, negative dithizone staining and no insulin secretion. Conclusion: Comparing to the Percoll density gradient centrifugation method, whole bone marrow method is a simple, economical and practical MSCs in vitro isolation method. 2. MSCs from adult human bone marrow can be induced into insulin-secreting cells in vitro by thioglycol, glutamine, EGF, bFGF and nicotinamide.