Department of Neurosurgery, Stanford School of Medicine, Stanford, California, United States of America.
PLoS One. 2012;7(7):e41120. doi: 10.1371/journal.pone.0041120. Epub 2012 Jul 17.
The use of human embryonic stem cells (hESCs) to repair diseased or injured brain is promising technology with significant humanitarian, societal and economic impact. Parkinson's disease (PD) is a neurological disorder characterized by the loss of midbrain dopaminergic (DA) neurons. The generation of this cell type will fulfill a currently unmet therapeutic need. We report on the isolation and perpetuation of a midbrain-specified self-renewable human neural stem cell line (hNSCs) from hESCs. These hNSCs grew as a monolayer and uniformly expressed the neural precursor markers nestin, vimentin and a radial glial phenotype. We describe a process to direct the differentiation of these hNSCs towards the DA lineage. Glial conditioned media acted synergistically with fibroblastic growth factor and leukemia inhibitory factor to induce the expression of the DA marker, tyrosine hydroxylase (TH), in the hNSC progeny. The glial-derived neurotrophic factor did not fully mimic the effects of conditioned media. The hNSCs expressed the midbrain-specific transcription factors Nurr1 and Pitx3. The inductive effects did not modify the level of the glutamic acid decarboxylase (GAD) transcript, a marker for GABAergic neurons, while the TH transcript increased 10-fold. Immunocytochemical analysis demonstrated that the TH-expressing cells did not co-localize with GAD. The transplantation of these DA-induced hNSCs into the non-human primate MPTP model of PD demonstrated that the cells maintain their DA-induced phenotype, extend neurite outgrowths and express synaptic markers.
利用人类胚胎干细胞（hESC）修复患病或受损的大脑是一项很有前景的技术，具有重大的人道主义、社会和经济影响。帕金森病（PD）是一种以中脑多巴胺能（DA）神经元丧失为特征的神经退行性疾病。这种细胞类型的产生将满足目前尚未满足的治疗需求。我们报告了从中分离和维持一种中脑特异性自我更新的人类神经干细胞系（hNSC）。这些 hNSC 作为单层生长，均匀表达神经前体细胞标志物巢蛋白、波形蛋白和放射状胶质表型。我们描述了一种将这些 hNSC 定向分化为 DA 谱系的方法。胶质细胞条件培养基与成纤维细胞生长因子和白血病抑制因子协同作用，诱导 hNSC 后代中 DA 标志物酪氨酸羟化酶（TH）的表达。胶质衍生的神经营养因子不能完全模拟条件培养基的作用。hNSC 表达中脑特异性转录因子 Nurr1 和 Pitx3。诱导作用没有改变谷氨酸脱羧酶（GAD）转录本的水平，GAD 转录本是 GABA 能神经元的标志物，而 TH 转录本增加了 10 倍。免疫细胞化学分析表明，表达 TH 的细胞与 GAD 不共定位。将这些诱导产生的 DA 的 hNSC 移植到非人类灵长类动物 MPTP 诱导的 PD 模型中，证明这些细胞保持其诱导的 DA 表型，延长神经突生长并表达突触标志物。
