Derivation of midbrain dopamine neurons from human embryonic stem cells
Derivation of midbrain dopamine neurons from human embryonic stem cells
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DOI：
10.1073/pnas.0404700101
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发表时间：
2004-08-24
影响因子：
11.1
通讯作者：
Studer, L
中科院分区：
文献类型：
--
作者：
Perrier, AL;Tabar, V;Studer, L
人类胚胎干（hES）细胞的定义是其广泛的自我更新能力和分化为人体任何细胞类型的潜力。将hES细胞用于发育生物学和再生医学的挑战是将广泛的分化潜力导向特定细胞命运的衍生。在神经系统内，hES细胞已显示在体外分化成神经祖细胞、神经元和星形胶质细胞。然而，据我们所知，任何给定的神经元亚型的选择性衍生尚未得到证实。在这里，我们描述的条件下，直接hES细胞进入中脑多巴胺能神经元的身份。神经外胚层分化触发的基质饲养细胞，然后通过定义的图案化分子，直接在体内中脑发育的顺序应用区域规范。通过关键转录因子（包括Pax 2、Pax 5和enrailed-1（En 1））的顺序表达、DA释放的测量、河豚毒素敏感性动作电位的存在以及酪氨酸羟化酶阳性突触末梢的电子显微镜可视化来监测中脑多巴胺（DA）神经元命运的进展。从三个独立的hES和两个猴胚胎干细胞系中证实了高产DA神经元衍生。无限数量的中脑DA神经元的可用性是探索hES细胞在帕金森病临床前模型中的潜力的第一步。该实验系统也为探索控制人中脑DA能神经元发育和功能的分子机制提供了强有力的工具。
Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their potential to differentiate into any cell type of the human body. The challenge in using hES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential toward the derivation of a specific cell fate. Within the nervous system, hES cells have been shown to differentiate in vitro into neural progenitor cells, neurons, and astrocytes. However, to our knowledge, the selective derivation of any given neuron subtype has not yet been demonstrated. Here, we describe conditions to direct hES cells into neurons of midbrain dopaminergic identity. Neuroectodermal differentiation was triggered on stromal feeder cells followed by regional specification by means of the sequential application of defined patterning molecules that direct in vivo midbrain development. Progression toward a midbrain dopamine (DA) neuron fate was monitored by the sequential expression of key transcription factors, including Pax2, Pax5, and engrailed-1 (En1), measurements of DA release, the presence of tetrodotoxin-sensitive action potentials, and the electron-microscopic visualization of tyrosine-hydroxylase-positive synaptic terminals. High-yield DA neuron derivation was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons is a first step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to probe the molecular mechanisms that control the development and function of human midbrain DA neurons.