University of California, Davis, 2921 Stockton Blvd., Room 1300, Sacramento, CA 95817, USA.
Stem Cell Rev Rep. 2012 Dec;8(4):1129-37. doi: 10.1007/s12015-012-9411-6.
microRNAs (miRNAs) are important modulators in regulating gene expression at the post-transcriptional level and are therefore emerging as strong mediators in neural fate determination. Here, by use of the model of human embryonic stem cell (hESC)-derived neurogenesis, miRNAs involved in the differentiation from neural stem cells (hNSC) to neurons were profiled and identified. hNSC were differentiated into the neural lineage, out of which the neuronal subset was enriched through cell sorting based on select combinatorial biomarkers: CD15-/CD29(Low)/CD24(High). This relatively pure and viable subpopulation expressed the neuronal marker β III-tubulin. The miRNA array demonstrated that a number of miRNAs were simultaneously induced or suppressed in neurons, as compared to hNSC. Real-time PCR further validated the decrease in levels of miR214, but increase in brain-specific miR7 and miR9 in the derived neurons. For functional studies, hNSC were stably transduced with lentiviral vectors carrying specific constructs to downregulate miR214 or to upregulate miR7. Manipulation of either miR214 or miR7 did not affect the expression of β III-tubulin or neurofilament, however miR7 overexpression gave rise to enhanced synapsin expression in the derived neurons. This indicated that miR7 might play an important role in neurite outgrowth and synapse formation. In conclusion, our data demonstrate that miRNAs function as important modulators in neural lineage determination. These studies shed light on strategies to optimize in vitro differentiation efficiencies to mature neurons for use in drug discovery studies and potential future clinical applications.
微小 RNA（miRNAs）是在后转录水平上调节基因表达的重要调节剂，因此作为神经命运决定的强介质而出现。在这里，通过使用人胚胎干细胞（hESC）衍生的神经发生模型，对涉及从神经干细胞（hNSC）分化为神经元的 miRNAs 进行了分析和鉴定。hNSC 分化为神经谱系，其中神经元亚群通过基于选择组合生物标志物的细胞分选进行富集：CD15-/CD29（低）/CD24（高）。这个相对纯净和有活力的亚群表达神经元标志物β III-微管蛋白。miRNA 阵列表明，与 hNSC 相比，神经元中同时诱导或抑制了许多 miRNAs。实时 PCR 进一步验证了 miR214 水平降低，但脑特异性 miR7 和 miR9 在衍生神经元中增加。对于功能研究，hNSC 被稳定转导带有特定构建体的慢病毒载体，以下调 miR214 或上调 miR7。miR214 或 miR7 的操作均不影响β III-微管蛋白或神经丝的表达，但 miR7 的过表达导致衍生神经元中突触素表达增强。这表明 miR7 可能在神经突生长和突触形成中起重要作用。总之，我们的数据表明 miRNAs 作为神经谱系决定的重要调节剂发挥作用。这些研究为优化体外分化效率以获得用于药物发现研究和潜在未来临床应用的成熟神经元提供了策略。
