Dmitry A Ovchinnikov, Nilay Y Thakar, Ernst J Wolvetang, Stem Cell Engineering Group, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane 4072, Queensland, Australia.
World J Stem Cells. 2012 Jul 26;4(7):71-9. doi: 10.4252/wjsc.v4.i7.71.
The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein.
Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression, flow cytometry, teratoma assays and embryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy.
Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival, motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein, mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette, driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripotency markers in the nucleus (POU5F1/OCT4, NANOG and SOX2) and on the cell surface (SSEA4, TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and, importantly, maintained robust mCherry expression throughout differentiation. The cell line was next adapted to single-cell passaging, rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces, quantification of cell attachment to nanoparticles and rapid estimation of cell survival.
The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation, making it a useful tool for bioengineering and in vivo tracking experiments.
生成并鉴定一个稳定表达红色荧光 mCherry 蛋白的人胚胎干细胞（hESC）系。
通过慢病毒转导，将一个组成型表达的人 EF-1α 启动子驱动的 mCherry 转基因整合到 MEL2 hESC 中。通过免疫荧光和流式细胞术评估红色荧光强度。通过免疫荧光多能性标志物表达、流式细胞术、畸胎瘤实验和类胚体分化后的逆转录聚合酶链反应（RT-PCR）来确定稳定转导的 hESC 的多能性。通过延时显微镜来定量细胞的迁移和存活能力。
稳定表达荧光蛋白的 hESC 是一种非常有用的工具，可用于体外和体内追踪细胞在分化前后在不同表面上的存活、迁移和细胞铺展情况。在此，我们描述了一个稳定表达红色荧光蛋白 mCherry 的 hESC 系（MEL2）的生成和鉴定。该系通过随机整合一个由泛表达的人 EF-1α 启动子驱动的荧光蛋白表达盒而产生。稳定转染的 MEL2-mCherry hESC 表达核内多能性标志物（POU5F1/OCT4、NANOG 和 SOX2）和细胞表面标志物（SSEA4、TRA1-60 和 TG30/CD9），并在长期（至少 35 代）培养中保持正常核型。MEL2-mCherry hESC 进一步在类胚体和畸胎瘤实验中容易分化为三个胚层的代表性细胞类型，重要的是，在整个分化过程中保持了强大的 mCherry 表达。该细胞系随后被适应于单细胞传代，使其与许多生物工程应用兼容，例如测量细胞在各种蛋白质修饰表面上的迁移和铺展能力、量化细胞对纳米颗粒的黏附以及快速估计细胞的存活率。
MEL2-mCherry hESC 系符合真正多能干细胞的标准，并在整个分化过程中保持红色荧光，使其成为生物工程和体内追踪实验的有用工具。
