Embryonic stem cell-derived Pitx3-enhanced green fluorescent protein midbrain dopamine neurons survive enrichment by fluorescence-activated cell sorting and function in an animal model of Parkinson's disease
Embryonic stem cell-derived Pitx3-enhanced green fluorescent protein midbrain dopamine neurons survive enrichment by fluorescence-activated cell sorting and function in an animal model of Parkinson's disease
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DOI：
10.1634/stemcells.2007-0996
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发表时间：
2008-06-01
期刊：
影响因子：
5.2
通讯作者：
Isacson, Ole
中科院分区：
文献类型：
--
作者：
Hedlund, Eva;Pruszak, Jan;Isacson, Ole
胎儿腹侧中脑（VM）和胚胎干（ES）细胞衍生的多巴胺神经元已成功地用于纠正帕金森病动物模型的行为反应。然而，来自胎儿VM细胞或ES细胞的移植物含有多种细胞类型，并且这些细胞中的大多数不是多巴胺神经元。ES细胞衍生的多巴胺神经元的分离和随后的移植将阐明这些神经元提供功能输入的能力，并进一步探索ES细胞用于治疗帕金森病的有效和安全的用途。为了实现这一目标，我们使用Pitx 3增强的绿色荧光蛋白（Pitx 3-eGFP）敲入小鼠囊胚衍生的胚胎干细胞（mES）细胞系和荧光激活细胞分选（FACS）来选择和纯化中脑多巴胺神经元。最初，在体外分化后评价完整Pitx 3-eGFP mES培养物的多巴胺能标志物谱。eGFP的表达与Pitx 3、Nurr 1、Engrailed-1、Lmx 1a、酪氨酸羟化酶（TH）、L-芳香族氨基酸脱羧酶（AADC）和囊泡单胺转运蛋白2（VMAT 2）的表达密切重叠，表明这些细胞具有中脑多巴胺神经元的特征。此外，有丝分裂后的Pitx 3-eGFP（+）多巴胺神经元（其在解离后构成培养物中所有活细胞的2%-5%）可以通过FACS高度富集至>90%纯度，并且这些分离的神经元是存活的、延伸的神经突，并且在体外维持多巴胺能谱。移植到6-羟基多巴胺损伤的大鼠表明，丰富的多巴胺能人口可以生存和恢复安非他明和阿朴吗啡诱导的功能，和移植物含有大量的中脑多巴胺神经元，它支配宿主纹状体。
Both fetal ventral mesencephalic (VM) and embryonic stem (ES) cell-derived dopamine neurons have been used successfully to correct behavioral responses in animal models of Parkinson's disease. However, grafts derived from fetal VM cells or from ES cells contain multiple cell types, and the majority of these cells are not dopamine neurons. Isolation of ES cell-derived dopamine neurons and subsequent transplantation would both elucidate the capacity of these neurons to provide functional input and also further explore an efficient and safer use of ES cells for the treatment of Parkinson's disease. Toward this goal, we used a Pitx3-enhanced green fluorescent protein (Pitx3-eGFP) knock-in mouse blastocyst-derived embryonic stem (mES) cell line and fluorescence-activated cell sorting (FACS) to select and purify midbrain dopamine neurons. Initially, the dopaminergic marker profile of intact Pitx3-eGFP mES cultures was evaluated after differentiation in vitro. eGFP expression overlapped closely with that of Pitx3, Nurr1, Engrailed-1, Lmx1a, tyrosine hydroxylase (TH), L-aromatic amino acid decarboxylase (AADC), and vesicular monoamine transporter 2 (VMAT2), demonstrating that these cells were of a midbrain dopamine neuron character. Furthermore, postmitotic Pitx3-eGFP(+) dopamine neurons, which constituted 2%-5% of all live cells in the culture after dissociation, could be highly enriched to >90% purity by FACS, and these isolated neurons were viable, extended neurites, and maintained a dopaminergic profile in vitro. Transplantation to 6-hydroxydopamine-lesioned rats showed that an enriched dopaminergic population could survive and restore both amphetamine- and apomorphine-induced functions, and the grafts contained large numbers of midbrain dopamine neurons, which innervated the host striatum.