Regenerative Bioscience Center, University of Georgia, Athens, Georgia 30602, USA.
Tissue Eng Part A. 2009 Nov;15(11):3621-34. doi: 10.1089/ten.tea.2009.0155.
Human embryonic stem cell-derived neural progenitors (NP) present an important tool for understanding human development and disease. Optimal utilization of NP cells, however, requires an enhanced ability to monitor these cells in vitro and in vivo. Here we report production of the first genetically modified self-renewing human embryonic stem cell-derived NP cells that express fluorescent proteins under constitutive as well as lineage-specific promoters, enabling tracking and monitoring of cell fate. Nucleofection, transfection, and lentiviral transduction were compared for optimal gene delivery to NP cells. Transduction was most efficient in terms of transgene expression (37%), cell viability (39%), and long-term reporter expression (>3 months). Further, the constitutive gene promoters, cytomegalovirus, elongation factor 1alpha, and ubiquitin-C, exhibited comparable silencing (20-30%) in NP cells over a 2-month period, suggesting their suitability for long-term reporter expression studies. Transduced NP cells maintained their progenitor state and differentiation potential, as demonstrated by expression of endogenous NP markers and neuronal markers after differentiation. We also detected reporter expression in astrocytes generated from NP cells transduced with an astrocyte-specific gene promoter, glial fibrillary acidic protein, demonstrating the usefulness of this approach. The genetically manipulated NP cells described here offer great potential for live cell-tracking experiments, and a similar approach can as well be used for expression of proteins other than reporters.
人胚胎干细胞衍生的神经祖细胞（NP）是理解人类发育和疾病的重要工具。然而，要优化 NP 细胞的利用，就需要增强在体外和体内监测这些细胞的能力。在这里，我们报告了第一批经过基因修饰的自我更新的人胚胎干细胞衍生的 NP 细胞的生产，这些细胞在组成型和谱系特异性启动子的控制下表达荧光蛋白，从而能够追踪和监测细胞命运。比较了核转染、转染和慢病毒转导在 NP 细胞中进行最佳基因传递的效果。就转导效率（37%）、细胞活力（39%）和长期报告基因表达（>3 个月）而言，转导效率最高。此外，在 2 个月的时间内，组成型基因启动子（巨细胞病毒、延伸因子 1alpha 和泛素 C）在 NP 细胞中表现出相似的沉默（20-30%），这表明它们适合于长期报告基因表达研究。转导的 NP 细胞保持其祖细胞状态和分化潜能，这可以通过分化后表达内源性 NP 标记物和神经元标记物来证明。我们还检测到了从 NP 细胞中转导了星形胶质细胞特异性基因启动子（胶质纤维酸性蛋白）的细胞中报告基因的表达，证明了这种方法的有用性。这里描述的经过基因修饰的 NP 细胞为活细胞追踪实验提供了巨大的潜力，并且类似的方法也可以用于表达除报告基因以外的蛋白质。
