Induction of midbrain dopaminergic neurons from primate embryonic stem cells by coculture with Sertoli cells
Induction of midbrain dopaminergic neurons from primate embryonic stem cells by coculture with Sertoli cells
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DOI：
10.1634/stemcells.2005-0409
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发表时间：
2006-07-01
期刊：
影响因子：
5.2
通讯作者：
Sasaki, Katsunori
中科院分区：
文献类型：
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作者：
Yue, Fengming;Cui, Li;Sasaki, Katsunori
本研究的目的是将灵长类胚胎干细胞（ES）与小鼠Sertoli细胞共培养后产生多巴胺能神经元。诱导3周后，免疫染色显示90% +/- 9%的菌落含有酪氨酸羟酶阳性（TH+）神经元，60% +/- 7%的微管蛋白β III阳性（Tuj III+）神经元为TH+。逆转录聚合酶链反应分析显示，sertoli诱导的神经元表达中脑多巴胺能神经元标志物，包括TH、多巴胺转运蛋白、芳香氨基酸脱羧酶（AADC）、TrkB和TrkC等受体以及转录因子NurrI和Lmx1b。在Sertoli细胞上分化的神经元中脑相关标志物Pax2、En1和AADC呈阳性，去甲肾上腺素能神经元标志物多巴胺- β -羟化酶呈阴性。这些支持细胞诱导的多巴胺能细胞在高K+去极化时释放多巴胺。支持细胞条件培养基中含有胶质细胞系来源的神经营养因子（GDNF），支持神经元分化。经抗gdnf抗体预处理后，Tuj III+菌落百分比降至14%。因此，GDNF在诱导灵长类胚胎干细胞形成多巴胺能神经元中起着重要作用。当移植到6-羟多巴胺治疗的帕金森病模型时，灵长类动物来源的多巴胺能神经元整合到小鼠纹状体中。移植两周后，存在存活的TH+细胞。这些TH+细胞存活了2个月。因此，胚胎干细胞与Sertoli细胞共培养的诱导方法为研究帕金森病的发病机制和移植提供了无限的灵长类细胞来源。
The aim of this study was to produce dopaminergic neurons from primate embryonic stem (ES) cells following coculture with mouse Sertoli cells. After 3 weeks of induction, immunostaining revealed that 90% +/- 9% of the colonies contained tyrosine hydroxylase-positive (TH+) neurons, and 60% +/- 7% of the tubulin beta III-positive (Tuj III+) neurons were TH+. Reverse transcription-polymerase chain reaction analyses showed that Sertoli-induced neurons expressed midbrain dopaminergic neuron markers, including TH, dopamine transporter, aromatic amino acid decarboxylase (AADC), receptors such as TrkB and TrkC, and transcription factors NurrI and Lmx1b. Neurons that had been differentiated on Sertoli cells were positive for Pax2, En1, and AADC, midbrain-related markers, and negative for dopamine-beta-hydroxylase, a marker of noradrenergic neurons. These Sertoli cell-induced dopaminergic cells can release dopamine when depolarized by high K+. Sertoli cell-conditioned medium contained glial cell line-derived neurotrophic factor (GDNF) and supported neuronal differentiation. After pretreatment with anti-GDNF antibody, the percentage of Tuj III+ colonies was reduced to 14%. Thus, GDNF contributed significantly to inducing primate ES cells into dopaminergic neurons. When transplanted into a 6-hydroxydopamine-treated Parkinson's disease model, primate-derived dopaminergic neurons integrated into the mouse striatum. Two weeks after transplantation, surviving TH+ cells were present. These TH+ cells survived for 2 months. Therefore, the induction method of coculture ES cells with Sertoli cells provides an unlimited source of primate cells for the study of pathogenesis and transplantation in Parkinson's disease.