| Parkinsons disease (PD) is a neurological disorder that usually affects old people. The pathological basis of PD is the degeneration of the dopamine neurons which project from substantia nigra to the striatum, resulting in the reduction of dopamine in the brain and a series of clinical manifestations such as shaking, hypermyotonia and cinesipathy. Currently, medication was used for treatment of PD but can not cure this disease.The experimental studies of cell transplantation for PD treatment have carried out for many years. In previous studies, neurons from midbrain or adrenal medulla cells used for transplantation didn't exert a good effect. Stem cells transplantation is an ideal way to treat Parkinson's disease, but it was difficult to find a suitable cell source. In recent years, neural stem cells are found to proliferate and differentiate into dopaminergic neurons, which are considered as the suitable donor cells to treat PD theoretically. However, the isolation of neural stem cells is difficult and hindered by ethics, which restricted the application of neural stem cells in the treatment of neurodegenerative disease.Because of their capacity of self-renewal and multi-lineage differentiation, bone marrow mesenchymal stem cells (BMSCs) have become an appealing donor graft source, leading to tremendous studies on their roles in cell transplantation for therapy of diseases and injuries. BMSCs can differentiate into neurons overcoming the germinal layer commitment under certain conditions. Another advantage of BMSCs is that these cells may avoid ethical and logistical hurdles in transplantation. However, there were no effect methods of inducing BMSCs differentiating into dopaminergic neurons up to now. The dopaminergic neurons project to the striatum. During the process of embryonic development, the neurons in the midbrain gradually project to the striatum and differentiate into functional dopaminergic neurons induced by the striatal microenvironment. Studies have shown that the striatum contains a variety of neurotrophic factors, which play important roles on the growth and development of dopaminergic neurons. There are in vivo and in vitro experiments in this study. Our study aimed (i) to test the hypothesis that striatal tissue extract can facilitate the differentiation of BMSCs into neural derivatives when they are cultured in vitro; (ii) to analyze the differentiation and distribution of BMSCs after intrastriatal transplantation.In transplantation, the transplanted cells need to be labeled and we can distinguish labeled cells from host tissue. Bis benzamide (BBZ) and 5-bromo-2-deoxyuridine (BrdU) were frequently used to label donor cells in previous studies, but the traditional cell labels of BBZ and BrdU can transfer to host cells, which might lead to inaccurate results. Therefore, we chose BMSCs isolated from green fluorescent protein (GFP) transgenic mouse (GFP-BMSCs) as donor cells in this study. The green fluorescence of GFP is activated without any substrates. With the help of GFP, the survival, differentiation and distribution of GFP-BMSCs were easily observed in the host brain.GFP-BMSCs isolated from GFP transgenic mouse were adherent cultured. Flow cytometry was used to identify the expression of CD29 and CD11b of the third generation of the BMSCs. The growth curve of the third generation cells was drawn. In vitro, the striatal tissue extract was added into the culture medium of GFP-BMSCs. Then, we analyzed the survival, differentiation and the cell-cycle changes of GFP-BMSCs compared with those in the normal culture medium through neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), tyrosine hydmxylase (TH) immunocytochemical staining and flow cytometry.In the in vivo experiment, we transplanted GFP-BMSCs into rat striatum, and observed the survival and distribution of GFP-BMSCs at Id,7d,14d,28d and 45d posttransplantation. Then the differentiation of transplanted GFP-BMSCs was examed through immunohistochemistry staining.The in vitro cultured GFP-BMSCs grew well. Cells appeared green under a fluorescent microscope. The result of the flow cytometry showed that GFP-BMSCs could express CD29 and almost not express CD11b, which illustrated the high purity of the cells. The growth curve suggested the good proliferation of the cells of third generation. Flow cytometry result demonstrated that majority of GFP-BMSCs of third generation both in the two groups were in GO/Gl phase, which indicated the good proliferation of GFP-BMSCs. Striatal tissue extract promoted the GFP-BMSCs convert from GO/G1 into S phase, namely, promoted the DNA replication and protein synthesis of the cells. Immunocytochemical staining analysis indicated that there were more NSE, GFAP and TH positive cells in experiment group (37.00±4.29%, 14.56±2.49% and 15.00±3.83%, respectively) than the control group(2.30±0.77%,0 and 0, respectively) (all p<0.01), namely, striatal tissue extract could promote the neural differentiation of GFP-BMSCs, epecially the differentiation into dopaminergic neurons.Under a fluorescence microscope, we could easily distinguish donor cells from host tissue and observe the survival and distribution of the transplanted GFP-BMSCs. GFP-BMSCs were found in the striatum, cortex, lateral cerebral ventricle and corpus callosum. In immunohistochemistry staining of brain sections, NSE, GFAP and TH-positive GFP-BMSCs were observed. The results demonstrated that GFP-BMSCs could differentiate into dopaminergic neurons after intrastriatal transplantation.Taken together, our results indicate that the striatal tissue extract and the striatal microenvironment can promote the neural differentiation of GFP-BMSCs, epecially the differentiation into dopaminergic neurons. |
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