Generation of Human Embryonic Stem Cell Reporter Lines Expressing GFP Specifically in Neural Progenitors
Generation of Human Embryonic Stem Cell Reporter Lines Expressing GFP Specifically in Neural Progenitors
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DOI：
10.1007/s12015-010-9159-9
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发表时间：
2010-09-01
影响因子：
4.8
通讯作者：
Cui, Wei
中科院分区：
文献类型：
--
作者：
Noisa, Parinya;Urrutikoetxea-Uriguen, Alai;Cui, Wei
谱系特异性人胚胎干细胞（hESC）报告系的产生将有助于实时监测活细胞的分化以及识别控制这些过程的因素。它还将使研究人员能够从异质分化的 hESC 后代中纯化特定的细胞群。在这里，我们报告了克隆衍生的巢蛋白-EGFP报告基因hESC系的产生，这些系在神经上皮特异性巢蛋白第二内含子增强子的控制下表达GFP。我们表明，nestin-EGFP hESC 报告系保留了未分化 hESC 的特征，能够在 hESC 培养条件下自我更新并分化为所有三个胚层的细胞。尽管在未分化状态下可检测到低水平，但巢蛋白-EGFP报告基因在分化后的神经祖细胞中表现出高表达。此外，转基因的表达仅局限于分化后的神经祖细胞。转基因的特异性表达由巢蛋白增强子中 POU 和 SOX 转录因子的协同结合基序决定。删除任一结合元件都会导致增强子/启动子活性显着降低。综上所述，nestin-EGFP 报告基因 hESC 系不仅对于研究 hESC 的神经分化过程非常有价值，而且对于富集其他细胞谱系的神经祖细胞也具有不可估量的价值。
Generation of lineage-specific human embryonic stem cell (hESC) reporter lines will facilitate the real time monitoring of differentiation in live cells and the identification of factors governing these processes. It will also enable researchers to purify specific cell populations from heterogeneous differentiated hESC progeny. Here we report the generation of clonally derived nestin-EGFP reporter hESC lines that express GFP under the control of the neuroepithelial specific nestin 2nd intron enhancer. We show that the nestin-EGFP hESC reporter lines retain the features of undifferentiated hESCs, are able to self-renew in hESC culture conditions and to differentiate into cells of all three germ layers. The nestin-EGFP reporter exhibited high expression in neural progenitor cells upon differentiation, although it is detectable at a low level in the undifferentiated state. Furthermore, the expression of the transgene is exclusively confined to the neural progenitors after differentiation. The specific expression of the transgene is determined by collaborative binding motifs of POU and SOX transcription factors in the nestin enhancer. Deletion of either of the binding elements resulted in a significant reduction of enhancer/promoter activity. Taken together, the nestin-EGFP reporter hESC lines are invaluable not only for the study of the neural differentiation process from hESCs but also for the enrichment of neural progenitor cells from other cell lineages.