Study On Directed Differentiation Of Bone Mesenchymal Stem Cells Into Neural-like Cells In Vitro

Symptoms of neurogenic bladder caused by spinal cord injury affect the quality of patient life severely, but there is no effective treatment currently, futhermore, most studies focused on the area of urodynamic and neurological damage. With the rapid development of stem cell research, not only stem cell transplantation has been used to treat the blood system cancer and autoimmune disease of nervous system, but also has achieved effective results, Even has been used in clinical. It is possible that the addition of the number of spinal nerves and the reduction of glial scar transplanted by stem cell together with tissue engineering, which will become the effective method of treatment of neurogenic bladder caused by spinal cord injury. However, suitable choice on seeding cell become important treatment in the construction of tissue engineering. All stem cells and pluripotent stem cells have a high degree of self-renewal ability and potential capability of differentiation, which is an important target cells in tissue engineering. Embryonic stem cells exist ethical issues and immune rejection, source of neural stem cells and schwann cell are limited, and cells derived inconvenience. Therefore , clinical application is difficult. BMSCs have the power ability of proliferation in vitro, moreover, can be differentiated into bone, fat, tendon and muscle in different physical and chemical environment, even can be differentiated into nerve tissue across embryos, which show the favorable prospect of research and application.ObjectiveUsed the whole bone marrow adherent method, BMSCs of SD rat were isolated, purified and amplified in vitro. To observe the structure of morphology and phenotypic identification, we conduct immunofluorescence dyeing and detection of flow cytometry. Biological characteristics of BMSCs were used to conduct analysis of immunocytochemistry, induction and identification of differentiation. then we explore potential ability of BMSCs which differentiate into neural-like cells. The experimental study provides an ideal seeding cells and a theoretical basis for the treatment of neurogenic bladder caused by spinal cord injury.Methods1. BMSCs of SD rat were extracted, isolated, purified and amplified by the whole bone marrow adherent method in vitro;2. We drew the growth curve of BMSCs, and conduct immunofluorescence dyeing for observation on the structure of BMSCs;3. The positive expression of cell surface antigens CD29, CD34 and CD105 were detected by flow cytometry;4. BMSCs were induced to differentiate into osteoblasts and nerve cell under the inducer of osteoblast and nerve cell conditions, moreover,were stained by alizarin red and nestin immunocytochemistry staining respectively;5. The neurospheres of induced formation were cultured and differentiated furtherly. To identify the formation of neurons and glial cells, BMSCs were stained by MAP-2, GFAP immunocytochemistry staining.Results1. BMSCs obtained by the whole bone marrow adherent method attachs the bottle after 24～36 hours, showed the growth of spindle whirlpool and the power ability of proliferation, nuclear-cytoplasmic ratio of BMSCs is large;2. Cell growth pass the potential adaptation period, the increased logarithmic phase and plateau phase, the whole cells show green fluorescent and good shape, nucleus was not stained fluorescence;3. Flow cytometry showed the positive expression of the 3～5 passage cell surface antigens CD29 and CD105,while CD34 expression was negative;4. Under the different inducer conditions, BMSCs can be differentiated into osteoblast and nerve-like cells, moreover, expressed the corresponding antigen;5. The neurospheres of induced formation furtherly were able to express neuronal markers MAP-2 or the glial marker GFAP.Conclusion1. The Whole bone marrow adherent method can separate BMSCs of high purity and obtain a large number of BMSCs adhered to the wall and owned the power ability of proliferation;2. BMSCs have more potential to differentiate, and can be differentiated into osteoblast and nerve-like cells in vitro;3. The neurospheres of induced formation can be differentiated into osteoblast and nerve-like cells futherly, BMSCs is an ideal seed cells in the transplantation of stem cell, which possess glorious prospects.