Rapid Differentiation of Human Embryonal Carcinoma Stem Cells (NT2) into Neurons for Neurite Outgrowth Analysis
Rapid Differentiation of Human Embryonal Carcinoma Stem Cells (NT2) into Neurons for Neurite Outgrowth Analysis
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DOI：
10.1007/s10571-011-9659-4
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发表时间：
2011-05-01
影响因子：
4
通讯作者：
Bicker, Gerd
中科院分区：
文献类型：
--
作者：
Tegenge, Million Adane;Roloff, Frank;Bicker, Gerd
来源于干细胞的人神经元可以用作体外模型来预测神经化学物质影响神经发育细胞过程（包括增殖、迁移和分化）的潜力。在这里，我们开发了一个模型，从充分表征的人胚胎癌干细胞（NT 2）分化人类神经元。NT 2细胞诱导分化成神经元表型后，2周的治疗与视黄酸在聚集体培养。巢蛋白阳性祖细胞迁移出NT 2聚集体并分化成表达β III-微管蛋白的神经元细胞。将NT 2细胞再培养7-14天导致表达β III-微管蛋白的细胞的百分比增加，产生了轴突标记物（Tau）和突触前蛋白（突触蛋白）阳性染色的长神经突。然后，我们问是否从NT 2细胞的神经突生长是由生物活性化学物质调制。由于cAMP/PKA途径已被广泛研究，在几个实验系统中作为神经突生长/再生的调节剂，我们在培养中使用cAMP/PKA途径的化学活化剂和抑制剂。腺苷酸环化酶激活剂毛喉素和cAMP的细胞可渗透类似物8-Br-cAMP增加了神经突承载细胞和神经突延伸的百分比。蛋白激酶A抑制剂H-89和Rp-cAMP的应用阻断了神经突的形成。总之，NT 2聚集体经历迁移、分化和神经突细化，并且可以用作分化人类神经元的模型以筛选神经化学物质并理解人类神经细胞发育的细胞机制。
Human neurons derived from stem cells can be employed as in vitro models to predict the potential of neurochemicals affecting neurodevelopmental cellular processes including proliferation, migration, and differentiation. Here, we developed a model of differentiating human neurons from well characterized human embryonal carcinoma stem cells (NT2). NT2 cells were induced to differentiate into neuronal phenotypes after 2 weeks of treatment with retinoic acid in aggregate culture. Nestin positive progenitor cells migrate out of NT2 aggregates and differentiate into beta III-tubulin expressing neuronal cells. Culturing the NT2 cells for an additional 7-14 days resulted in increased percentage of beta III-tubulin expressing cells, elaborating a long neurite that positively stained for axonal marker (Tau) and presynaptic protein (synapsin). We then asked whether neurite outgrowth from NT2 cells is modulated by bioactive chemicals. Since the cAMP/PKA pathway has been widely investigated as a regulator of neurite outgrowth/regeneration in several experimental systems, we used chemical activators and inhibitors of cAMP/PKA pathway in the culture. The adenylyl cyclase activator, forskolin, and cell-permeable analog of cAMP, 8-Br-cAMP increased the percentage of neurite bearing cells and neurite extension. Application of the protein kinase A inhibitors, H-89 and Rp-cAMP, blocked neurite formation. Taken together, NT2 aggregates undergo migration, differentiation, and neurite elaboration and can be used as a model of differentiating human neurons to screen neurochemicals and to understand cellular mechanisms of human nerve cell development.