Generation of dopaminergic neurons in the adult brain from mesencephalic precursor cells labeled with a nestin-GFP transgene
Generation of dopaminergic neurons in the adult brain from mesencephalic precursor cells labeled with a nestin-GFP transgene
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DOI：
10.1523/jneurosci.21-11-03895.2001
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发表时间：
2001-06-01
影响因子：
5.3
通讯作者：
Okano, H
中科院分区：
文献类型：
--
作者：
Sawamoto, K;Nakao, N;Okano, H
有朝一日，中脑前体细胞可能会为帕金森病的治疗提供多巴胺能神经元。然而，从中脑前体产生多巴胺能神经元一直很难追踪，部分原因是还没有一种合适的方法来识别中脑室带前体。为了可视化和分离混合种群中的中脑前体细胞，我们使用了携带绿色荧光蛋白(GFP)基因的转基因小鼠和大鼠，并在Nestin增强子的控制下进行了研究。巢蛋白驱动的绿色荧光蛋白在中脑室区域被检测到，并与神经前体细胞的特异性标志物共定位。此外，流式细胞仪的数据显示，脑室带细胞的细胞表面标志Prominin/CD133在这些GFP阳性(GFP(+))细胞中特异表达。经荧光激活细胞分选后，GFP(+)细胞在体外增殖，表达前体细胞标志，但不表达神经元标志。利用克隆球形成实验，我们发现这个分选群体富含多能前体细胞，可以分化为神经元和神经胶质细胞。重要的是，许多从Nestin-GFP分类的中脑前体细胞产生的神经元在体外形成了多巴胺能表型。最后，将Nestin-GFP(+)细胞移植到帕金森病大鼠模型的纹状体内。溴脱氧尿苷-酪氨酸羟化酶双标记显示，移植细胞在宿主纹状体内产生了新的多巴胺能神经元。植入的细胞能够恢复宿主纹状体中的多巴胺能功能，这是通过一项行为测量来评估的：从苯丙胺诱导的旋转中恢复。综上所述，这些发现表明，从胚胎腹侧中脑获得的前体细胞可以产生能够恢复化学去神经支配的成年纹状体功能的多巴胺能神经元。
Mesencephalic precursor cells may one day provide dopaminergic neurons for the treatment of Parkinson's disease. However, the generation of dopaminergic neurons from mesencephalic precursors has been difficult to follow, partly because an appropriate means for recognizing mesencephalic ventricular zone precursors has not been available. To visualize and isolate mesencephalic precursor cells from a mixed population, we used transgenic mice and rats carrying green fluorescent protein (GFP) cDNA under the control of the nestin enhancer. nestin-driven GFP was detected in the mesencephalic ventricular zone, and it colocalized with specific markers for neural precursor cells. In addition, data from flow-cytometry indicated that Prominin/CD133, a cell-surface marker for ventricular zone cells, was expressed specifically in these GFP-positive (GFP(+)) cells. After sorting by fluorescence-activated cell sorting, the GFP(+) cells proliferated in vitro and expressed precursor cell markers but not neuronal markers. Using clonogenic sphere formation assays, we showed that this sorted population was enriched in multipotent precursor cells that could differentiate into both neurons and glia. Importantly, many neurons generated from nestin-GFP-sorted mesencephalic precursors developed a dopaminergic phenotype in vitro. Finally, nestin-GFP(+) cells were transplanted into the striatum of a rat model of Parkinson's disease. Bromodeoxyuridine-tyrosine hydroxylase double-labeling revealed that the transplanted cells generated new dopaminergic neurons within the host striatum. The implanted cells were able to restore dopaminergic function in the host striatum, as assessed by a behavioral measure: recovery from amphetamine-induced rotation. Together, these findings indicate that precursor cells harvested from the embryonic ventral mesencephalon can generate dopaminergic neurons able to restore function to the chemically denervated adult striatum.