| Objective Within the bone marrow stroma there exists a subset of nonhematopoietic stem cells referred to as marrow stromal cells or mesenchymal stem cells. Marrow stromal cells (MSCs) are a group of cells with highly capability of self-renew and potential of multilineage differentiation, these properties make them present a promising prospect for clinical practice and be the focuses in the study of neuroscience. Here we observe the biological features of MSCs and explore the protocols to inducing MSCs into neuron in vitro. We also observed the survival and migration after transplantation of induced MSCs into focal crebral ischemic rats. We also primal explored the possible mechanism of MSCs differentiation. The purpose of this study was to provided theoretic basis and new therapy methods for repairing injury of central nervous system with transplantation of MSCs. Methods1. The bone marrow was aspirated from the femurs of rats and MSCs was isolated by gradient centrifugation in percoll. The mononuclear cells were collected and cultured in vitro, and the adherent cells were kept. The higher purity of MSCs was obtained by repeatedly removal of nonadherent cells. They biological features were observed under phase contrast microscope and their growth curves were drawn. 2. The effects of different dosage of SF on growth and proliferation of MSCs were assayed by MTT assay and the optimal dosage was chosen. 3. In this study, MSCs between passages 3 and 6 were induced by sodium ferulate(SF) and β-mercaptocthand(β-ME). The morphological changes of induced MSCs were observed in different culture period and neuronal specific markers, such as microfilament (NF), Nestin, NSE and GFAP were detected by indirect immunocytochemistry. 4. Different dosage of PD98059, a specific inhibitor of MAPK (mitogen-activate protein kinase), was added into medium, the changes of MSCs morphologic and the expressing of neurons specific markers were observed.5. Focal cerebral ischemia was induced in rats by occlusion of the right middle cerebral artery (MCAo) using an intraluminal nylon thread. MSCs (about 2×106)which were pre-labeled with BrdU and induced by SF in vitro were implanted into the lesioned hemisphere within 2h after MACo in rats. The survival and migration of implanted MSCs in ischemic brains were observed by detecting BrdU positive cell with immunohistochemistry.Results 1. A population of relative homogenous MSCs was isolated from rat bone marrow by a density gradient centrifugation. The cultured MSCs attached to the plastic surface, spread and grew as fibroblastic cells that developed into colonies in primary culture. The adherent fibroblast-shaped cells came to confluence by days 10-14. The double time of MSCs was about 72h. 2. The cultured medium with 0.5mg/ml or 1mg/ml has no significantly effects on the growth and proliferation of MSCs, the survival rates of MSCs are 95% and 94% respectively; in contrast the medium containing 2mg/ml or 3mg/ml sodium ferulate has great effects on the growth and proliferation of MSCs, the survival rates of MSCs are 50% and 20% respectively after cultured for 7days. 3. The MSCs exhibit a neuron morphology after induced by SF for 6h. The refractile cells bodies extended long processes, termination in typical growth cone; 24h later, the complex networks were formed by clusters of MSCs-derived neurons. The differentiation cells expressed nestin, characteristic of neuronal precursor stem cells, at 6h, but the trait was undetectable at 24h. In contrast, expression of NF,NSE and GFAP, markers of mature neurons, persisted from 6h to 7days. Progressive transition of MSCs to a neuronal phenotype coincided with increased expression of NSE and GFAP; NSE and GFAP positive cells were found in MSCs induced by 1mg/ml SF for 3 days and the positive rate were 67±3.5% and 39±1.8%. Neuron morphology changes were apparent in MSCs which were exposure toβ-ME for 1h. Responsive cells progressively assurmed neuronal morphological characteristics over the first 3h. |
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