In Vitro Differentiation Of Adult Rat Bone Marrow Mesenchymal Stem Cells Into Insulin-producing Cells

Islet transplantation as a potential treatment for diabetes has been investigated extensively over the past 10 years. Such an approach, however, will always be limited mainly because it is difficult to obtain sufficiently large numbers of purified islets from cadaveric donors. One alternative to organ or tissue transplantation is to use a renewable source of cells. Stem cells are clonogenic cells capable of both self-renewal and multilineage differentiation into any type of cell and to be genetically modified in vitro, thus providing cells which can be isola-ted and used for transplantation. Recent studies have given well defined differentiation protocols, which can be used to guide stem cells into specific cell lineages as neurons, cardiomytocyte and insulin-secreting cells.Bone marrow mesenchymal stem cells ( MSCs ) have a pluripotent ability to differentiate a variety of cell lieages in vitro. A number of protocols have been described to induce neuronal cells, adipocytes, smooth muscle myoctyes, skeletal muscles myocytes and cartilage cells from MSC. But the production of insulin-secreting cells from MSC has not been demonstrated . We present evidence that relative purified adult rat MSC can differentiate into insulin producing cells, when cocultured with pancreatic cells of embryonic rat.Methods1. MSCs isolation and in vitro cultures.a. Euthanize adult rat ( age 3 month) using cervical dislocation. In the sterile condition take the thighbone and shankbone of adult rat ( age 3 month ) asprimary culture.b. Immunocytochemistry identification of the expressions of CD45and CD902. Pancreatic cells (PCs) isolation and in vitro culturea. In the sterile condition take pancreatic of rat ( age 1 week) as primary culture.b. DTZ stain identification of pancreatic B cell.3. Coculture of PCs and MSCs and identification of the cocultued cella. Reverse Transcription and PCR identification of the expression of insulin and PDX-1b. DTZ stain identification of cocultured cells.c. Immunocytochemistry and immunofluorescence identification of the expressions of nestin and insulin.ResultsIsolate and culture the MSC of the adult rat, Immunocytochemistry showed that most of the fifth primary MSC express CD45 (-) CD90( + ). Cocultured cell was stained crimson red by DTZ. RT-PCR showed that the cocultured cell express insulin and PDX-1, Immunocytochemistry show the express of insulin and the same result of immunofluorescence.ConclusionsMSCs cultured in vitro are potent to differentiate into pancreatic islet-like tissue and they can differentiate into pancreatic B cells.