In Vitro Transdifferentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Insulin-secreting Cells

【Objective】To investigate the possibility of bone marrow mesenchymal stem cells to transdifferentiate into insulin-secreting cells, and to demonstrate whether these islet-like cells can generate two hormones: insulin and glucagon.【Methods】BMSCS were isolated from tibia or thighbone of 6-8 weeks normal SD rat, purified on the basis of their ability to adhere to the matrix, and expanded through their self-renewal. Two-step strategy was adopted, BMSCS were induced to generate nestin-positive cells under differentiation culture medium 1 (added 20 ng. ml-1 bFGF, 10ng. ml-1 EGF and 2%B27 in DMDM/F12 medium) in step one; Nestin-positive cells were differentiated and developed into insulin-secreting cells or islet progenitors under complex culture medium 2 (added 10 ng. ml-1 Betacellulin, 10 ng. ml-1 HGF, 10 ng. ml-1 Activitin A, 10 mmol. l-1 nicotide and 2 % B27 serum-free in HG-DMEM medium) in step two. The differentiated cell masses were tested by Dithiazone staining; Immunocytochemistry was carried out using standard protocols in which the cells were incubated with Rb-Rat Insulin monoclone antibody, Mouse-Human Glucogan polyclone antibody and Rb-Rat nestin monoclone antibody, assessed with a Zeiss microscope. Insulin secretion in response to insulin secretagogues (Glucose) was assayed by radioimmunoassay (RIA).【Result】The results of BMSCs surface protein expressing (P6) is: CD45 (-),CD29(+)and CD90(+), and the percent of purification is 99.88%. After 3 days, nestin protein, a marker shared with neural stem cells and adult pancreatic stem cells, were detected of BMSCs ,. Dithiazone staining was positive (bright orange). The differentiated cell masses were containing insulin and glucagon by Immunocytochemistry. RIA demonstrated that these cell...