Mamidi Murali Krishna, Pal Rajarshi, Bhonde Ramesh, Zakaria Zubaidah, Totey Satish
Stempeutics Research Malaysia Sdn. Bhd., Kuala Lumpur, Malaysia.
J Biomol Screen. 2010 Jul;15(6):630-43. doi: 10.1177/1087057110370211. Epub 2010 Jun 8.
Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.
评估基因表达谱的技术，包括实时定量PCR、TaqMan低密度芯片和灵敏度足够高的cDNA微阵列，是监测人类胚胎干细胞（hESC）培养的有效方法。然而，由于成本高、周转时间长以及需要高度专业化的技术专长，这些高通量检测大多用途有限。因此，缺乏用于hESC常规筛选的快速、经济高效、稳健且灵敏的方法。hESC培养的一个关键要求是维持均匀的未分化状态，并通过显示代表所有胚层（包括外胚层、中胚层和内胚层）的基因标志物的表达来确定其分化能力。为了量化hESC在通过胚状体（EB）形成进行增殖、扩增和分化过程中基因表达的调控，作者开发了一种简单、快速、廉价且确定的多标志物半定量多重RT-PCR（mxPCR）平台技术。在测试的15种基因引物中，4种是多能性标志物，组成第1组，来自外胚层、中胚层和内胚层的各3种谱系特异性标志物分别组合为第2至4组。作者发现这4组不仅在确定hESC的相对分化方面有效，而且易于重复。在本研究中，他们使用HUES-7细胞系对该技术进行标准化，随后用HUES-9、NTERA-2和小鼠胚胎成纤维细胞进行了验证。这种单反应mxPCR检测方法具有灵活性，通过选择合适的报告基因，可设计用于在常规维持和定向分化过程中对不同hESC系进行表征。
