Yang HongNa, Wang Jing, Wang Feng, Liu XiaoDun, Chen Heng, Duan WeiMing, Qu TingYu
Department of Critical-Care Medicine, Qilu Hospital of Shandong University, Shandong UniversityJinan, China; Department of Psychiatry, College of Medicine, University of Illinois at ChicagoChicago, IL, USA.
Department of Critical-Care Medicine, Qilu Hospital of Shandong University, Shandong University Jinan, China.
Front Neural Circuits. 2016 Apr 20;10:29. doi: 10.3389/fncir.2016.00029. eCollection 2016.
Transplantation of dopaminergic (DA) neurons is considered to be the most promising therapeutic strategy for replacing degenerated dopamine cells in the midbrain of Parkinson's disease (PD), thereby restoring normal neural circuit function and slow clinical progression of the disease. Human neural stem cells (hNSCs) derived from fetal forebrain are thought to be the important cell sources for producing DA neurons because of their multipotency for differentiation and long-term expansion property in cultures. However, low DA differentiation of the forebrain-derived hNSCs limited their therapeutic potential in PD. In the current study, we explored a combined application of Pramipexole (PRX), bone morphogenetic proteins 7 (BMP-7), and growth factors, including acidic fibroblast factor (aFGF), forskolin, and phorbol-12-myristae-13-acetate (TPA), to induce differentiation of forebrain-derived hNSCs toward DA neurons in cultures. We found that DA neuron-associated genes, including Nurr1, Neurogenin2 (Ngn2), and tyrosine hydroxylase (TH) were significantly increased after 24 h of differentiation by RT-PCR analysis (p < 0.01). Fluorescent examination showed that about 25% of cells became TH-positive neurons at 24 h, about 5% of cells became VMAT2 (vascular monoamine transporter 2)-positive neurons, and less than 5% of cells became DAT (dopamine transporter)-positive neurons at 72 h following differentiation in cultures. Importantly, these TH-, VMAT2-, and DAT-expressing neurons were able to release dopamine into cultures under both of the basal and evoked conditions. Dopamine levels released by DA neurons produced using our protocol were significantly higher compared to the control groups (P < 0.01), as examined by ELISA. Our results demonstrated that the combination of PRX, BMP-7, and growth factors was able to greatly promote differentiation of the forebrain-derived hNSCs into DA-releasing neurons.
多巴胺能（DA）神经元移植被认为是替代帕金森病（PD）中脑退化多巴胺细胞最有前景的治疗策略，从而恢复正常神经回路功能并减缓疾病的临床进展。源自胎儿前脑的人类神经干细胞（hNSCs）因其在培养中的多能分化能力和长期扩增特性，被认为是产生DA神经元的重要细胞来源。然而，前脑来源的hNSCs的低DA分化限制了它们在PD中的治疗潜力。在本研究中，我们探索了普拉克索（PRX）、骨形态发生蛋白7（BMP-7）和生长因子（包括酸性成纤维细胞因子（aFGF）、福斯可林和佛波醇-12-肉豆蔻酸酯-13-乙酸酯（TPA））的联合应用，以诱导培养中的前脑来源hNSCs向DA神经元分化。我们发现，通过RT-PCR分析，在分化24小时后，与DA神经元相关的基因，包括Nurr1、神经生成素2（Ngn2）和酪氨酸羟化酶（TH）显著增加（p < 0.01）。荧光检查显示，在培养物中分化72小时后，约25%的细胞在24小时时成为TH阳性神经元，约5%的细胞成为VMAT2（血管单胺转运体2）阳性神经元，不到5%的细胞成为DAT（多巴胺转运体）阳性神经元。重要的是，这些表达TH、VMAT2和DAT的神经元能够在基础和诱发条件下将多巴胺释放到培养物中。通过ELISA检测，与对照组相比，使用我们的方案产生的DA神经元释放的多巴胺水平显著更高（P < 0.01）。我们的结果表明，PRX、BMP-7和生长因子的组合能够极大地促进前脑来源的hNSCs分化为释放DA的神经元。
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