Keyoung H M, Roy N S, Benraiss A, Louissaint A, Suzuki A, Hashimoto M, Rashbaum W K, Okano H, Goldman S A
Department of Neurology and Neuroscience, Cornell University Medical College and New York Presbyterian Hospital, New York, NY 10021, USA.
Nat Biotechnol. 2001 Sep;19(9):843-50. doi: 10.1038/nbt0901-843.
Neural stem and precursor cells reside in the ventricular lining of the fetal forebrain, and may provide a cellular substrate for brain repair. To selectively identify and extract these cells, we infected dissociated fetal human brain cells with adenoviruses bearing the gene for green fluorescence protein (GFP), placed under the control of enhancer/promoters for two genes (nestin and musashi1) that are expressed in uncommitted neuroepithelial cells. The cells were then sorted by fluorescence-activated cell sorting (FACS) on the basis of E/nestin- or P/musashi1-driven GFP expression. Both P/musashi1:hGFP- and E/nestin:EGFP-sorted cells were multipotent: limiting dilution with clonal expansion as neurospheres, in tandem with retroviral lineage analysis and xenograft to E17 and P0-2 rat forebrain, revealed that each phenotype was able to both self-renew and co-generate neurons and glia. Thus, fluorescent genes placed under the control of early neural promoters allow neural stem cells to be specifically targeted, isolated, and substantially enriched from the fetal human brain.
神经干细胞和前体细胞存在于胎儿前脑的脑室衬里中，可能为脑修复提供细胞底物。为了选择性地识别和提取这些细胞，我们用携带绿色荧光蛋白（GFP）基因的腺病毒感染解离的胎儿人脑细胞，该基因置于在未分化神经上皮细胞中表达的两个基因（巢蛋白和神经母细胞标志物1）的增强子/启动子控制之下。然后根据E/巢蛋白或P/神经母细胞标志物1驱动的GFP表达，通过荧光激活细胞分选（FACS）对细胞进行分选。P/神经母细胞标志物1:hGFP分选细胞和E/巢蛋白:EGFP分选细胞都是多能的：以神经球形式进行有限稀释并克隆扩增，同时进行逆转录病毒谱系分析以及移植到E17和P0 - 2大鼠前脑，结果显示每种表型都能够自我更新并共同产生神经元和神经胶质细胞。因此，置于早期神经启动子控制之下的荧光基因能够使神经干细胞被特异性靶向、分离并从胎儿人脑中大量富集。
