| Parkinson’s disease (PD) is a neurodegenerative disorder in the elderly which is characteristic by loss of dopaminergic neurons and reduced dopamine synthesis in the substantia nigra. The main clinical symptoms of PD include resting tremor, rigidity, bradykinesia and postural instability. Currently, the treatments for PD are mainly focusing on pharmaceutics administration and neural surgeries, however, the existence therapies mostly confined to relieve symptoms but not fundamentally restore the lesion side function and loss of efficacy with disease progression. Therefore, a therapy method which is safe but also fundamentally to restore the function of the nigrostriatal system and can greatly ease the treatment of their symptoms is critical in clinical treatment of PD.It is a new idea that used stem cells for PD therapy. In this study, we induced genetically modified human umbilical cord mesenchymal stem cells (hUC-MSCs) into dopaminergic neurons and transplanted into the brain corresponding nuclei in the Parkinson’s disease rhesus monkey model. The LIM homeobox transcription factors1alpha (Lmxla) is sufficient and required to trigger midbrain dopamine (mDA) neurons differentiation. Neurturin (NTN) is a potent trophic factor for dopaminergic neurons. Based on the theory, we chose NTN and Lmxla as induction and protection genes respectively. The two genes were delivered into hUC-MSCs by recombinant adenovirus and combined with Lmxla regulatory factor-sonic hedgehog factor and other inductor to improve the efficiency of inducing. Then those induced cells were implanted into the striatum and substantia nigra of MPTP lesioned hemi-parkinsonian rhesus monkeys.The result shown that hUC-MSCs were successfully isolated from the umbilical cord and were negative for CD45. CD34and HLA-DR, but were positive for CD44, CD49d and CD29. After those cells were infected with recombinant adenovirus, RT-PCR result shows that both Lmx1α and NTN genes were transcribed in hUC-MSCs. We also observed that the exogenous were highly expressed in hUC-MSCs from immunofluorescence and western bolt. Experiments in vitro have proved that secretion NTN could maintain the survival of rat fetal midbrain dopaminergic neurons. We also explored the best exogenous and endogenous inducing factors to improve the efficiency and intracellular reprogramming to dopaminergic neurons differentiation. The mature neurons specific gene TH, Pitx3, EN was transcripted in differentiated cells detected by Real-time PCR and the immunofluorescence results prove that neurons specific protein TH,β-tubulinⅢ, NSE, Nestin, MAP-2was expressed in those differentiated cells. These results indicated that hUC-MSCs were able to differeniate into dopaminergic neuron-like cells. To confirm the physiological activity and therapy effect in vivo of induced neuron-like cells, we transplanted the cells labeled BrdU into substantia nigra (SN), putamen (Pu) and caudate nucleus (Cd) in Parkinson’s disease monkey model. First, we detected the timeliness and effectiveness of the models for six months to confirm that there is no spontaneous recovery in models, and then the transplantation therapy was carried out. The groups were including normal monkeys, control group with HBSS injection and experimental group with induced cells injection. We located the APO. SN, Pu and Cd nucleus area by magnetic resonance imaging (MRI) before transplation. Then we injected the induced dopaminergic neurons-like cells from hUC-MSCs labeled with BrdU were transplanted into the substantia nigra and striatum corresponding nuclei of the PD rhesus models by stereotactic injection technique. After the behavior, physiological recovery and dopamine transporter imaging evaluation, the results indicate that the symptoms of the monkey in cells therapy were greatly improved. After behavior evaluation, the monkeys were sacrificed with the method of euthanasia. HE, TH and BrdU immunohistochemistry results were shown that the grafted dopaminergic neuron-like cells were surviving in the lesioned side substantia nigra of the cell therapy group and the cells were derived from induced hUC-MSCs.Our data indicated that Lmxla played an important role in promoting hUC-MSCs differentiation. With the NTN nutrition protected and signaling molecules such as sonic hedgeho (SHH) and the fibroblast growth factor (FGF-8) induced hUC-MSCs was able to differentiate into dopaminergic neuron-like cells. On the one hand, transplanted dopaminergic neural-like cells are capable of replacing lost neurons and restoring the lost net work of striatal dopaminergic nerve terminals in PD.On the other hand, transplanted dopaminergic neural-like cells are capable of promoting the differentiation potential and providing trophic effects on endogenous neural stem cells probably through their continuous NTN and nutritional factors secretion. The furtuer secretion of NTN by induced DA neurons could provide neurons nutritional protection on the cells differentiated by induced cells and neural stem cells and form a virtuous circle. In this study, we induced hUC-MSCs into dopaminergic neuron-like cells in vitro and transplanted these cells in the early of Parkinson’s disease. These neuron-like cells could perform the physiological functions of dopaminergic neurons and may play a therapeutic role to ameliorate the symptoms of PD. The finding from this study may provide the basis for developing novel cell therapy for PD in future. |
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