Therapeutic Study Of Bone Marrow Mesenchymal Stem Cells On Rat Models Of Parkinson's Disease

Parkinson's disease(PD) is one of extrapyramidal diseases, characterized by selective degeneration of dopaminergic neurons in substantia nigra projecting to neostriatum. The capacity of dopamine from substantia nigra to striatum is decreased and symptoms are induced including paralysis agitans, myotonia, bradykinesia and so on. Intrastriatal grafting of dopamine-secreting cells from neural and paraneural tissues, such as fetal mesencephalon or adrenal medulla, has been reported to result in amelioration of the motor syndrome in PD patients and also in animal models. But moral problem and immunological rejection limited its use. So the most ideal transplantation method is: the grafts can keep their viability in vivo for a long time to produce dopamine, secrete all kinds of neurotrophins to improve the circumstances in graft and prevent progressive deterioration of PD. The aim of our study is to explore whether bone marrow mesenchymal stem cells(MSCs) can be induced to differentiate into neuron-like cell by different culture conditions. We studied Monoamine Neurotransmitters levels in brain slice co-culture with MSCs and the protection effect of MSCs to dopaminergic neuron in brain slice against l-methyl-4-phenylpyridinnium(MPP~+) toxicity. We also explored whether MSCs could alleviate the neurological deficits in PD rats and investigate the changes in magnetic resonance imaging (MRI) after transplantation.Part 1 Research on neuro-glial differentiation of MSCs by different culture conditionsAim: to explore whether MSCs can be induced to differentiate into neural-like cell by different culture conditionsMethod: MSCs obtained from adult bone marrow were cultured, isolated and purified. Cells were cultured in the following tissue cultue medium: DMEM+FBS;Neurobasal+B27;DMEM/F12+EGF+bFGF; co-cuture with neuron in absence of direct cell contact; co-culture with brain slice in presence of direct cell contact. The phase-contrast microscope and the technique of immunocytofluorescencewere applied to identify the differentiated MSCs. Meanwhile RT-PCR was performedto detect some genes in MSCs.Results: After 14 days co-culturing with neuron and brain slice, MSCs had alreadythe typical morphological neuron traits.the corn-like or spherical cell bodies and longprocesses and expressed several neuro-glial specific markers byimmuncocytochemistry and RT-PCR.Conclusion: Co-culture system was effective to induce the neuro-glial phenotype.Part 2 Influences on the monoamine neurotransmitter and dopaminergic neuron by co-culture bone marrow mesenchymal stem cell onto brain slice induced by l-methyl-4-phenyIpytidinium(MPP~+)Aim: To Study Monoamine Neurotransmitters levels in brain slice co-culture with MSCs and the protection effect of MSCs to dopaminergic neuron in brain slice against MPP+ toxicity.Method: The models of PD in vitro from newborn rats were established. MSCs obtained from adult bone marrow were cultured, isolated and purified. MSCs were co-cultured with brain slice. Immolunohistochemical technique, electron microscope, propidium iodide fluorescence were used to observe the change of neurons. Dopaminehydrochloride(DA), 3, 4—Dihydroxyphenylacetic acid (DOPAC) and Homovanillic acid (HVA) both in the tissue and culture medium were detected under high performance liquid chromatography with electrochemical detector(HPLC). Results: In the MPP~+ group, the neurites grew slowly and sparsely. Dead cells were found in all regions. In the co-culture group the neuritis grew densely, only a few cells were dead, the number of TH-stained neurons were increased and the structure of organellae looked normal. MPTP metabolite MPP~+ induced depletion of dopamine and its metabolites significantly. However, no significant changes of DA, DOPAC, HVA levels were detected in co-culture group.Conclusion: We suggest that MSCs may protect dopaminergic neurons against MPP~+ toxicity. it can be used for cell transplantation therapy to Parkinson's disease.Aim: To investigate the changes in magnetic resonance imaging (MRI) and curative effects after the transplantation of superparamagnetic iron oxide (SPIO) labeled MSCs in the treatment of PD models.Method: MSCs from adult bone marrow of SD rats were cultured, isolated and purified. Labeled MSCs was achieved with the magnetic resonance contrast agent SPIO, and Lipofectamine was used as transfection agent to achieve high transfer efficiencies. MSCs are also labed by BrdU. SPIO-labeled MSCs or BrdU labeled MSCs were transplanted to the right striatal area of 6-OHDA PD rat models. MR scanning was performed at 1, 2, 4, 6 weeks following transplantation, meanwhile, rotational behavior was assessed in each group. Brain frozen sections were carried out immunofluorescene double staining. Electron microscope was used to detect the ultra structure of MSCs.Results: SPIO particles were clearly visible with Prussian blue staining in cells. In vivo tracking magnetic resonance images especially T2* gradient echo showed that a dark signal appeared in the transplanted area, the dark signal area became larger as the transplanted times went on. Histochemical staining of brain tissue also shows that SPIO labeled MSCs were moved from the transplanted points to the nearby areas. Neuron-like cells were detected by Electron microscope. Some BrdU/Nestin -positive cells could be found in the implanted region of BrdU-labebed MSCs at 4 week after transplantation, but disappeared at 12 week. At this time some BrdU/MAP 2-positive cells appeared except BrdU/TH-positive cells.Conclusion: SPIO particles could label MSCs effectively, and MRI detection of SPIO labeled cells was a promising method and novel approach to analyse the MSCs following transplantation to PD models.