La Spada Alberto, Baronchelli Simona, Ottoboni Linda, Ruffini Francesca, Martino Gianvito, Convertino Nunzia, Ntai Aikaterini, Steiner Tobias, Biunno Ida, De Blasio Andrea
Institute of Genetic and Biomedical Research, National Research Council (IRGB-CNR), Milan, Italy (ALS, SB, IB).
Institute of Experimental Neurology (INSpe), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy (LO, FR, GM).
J Histochem Cytochem. 2016 Dec;64(12):739-751. doi: 10.1369/0022155416673969. Epub 2016 Oct 26.
In the past decade, tissue microarray (TMA) technology has evolved as an innovative tool for high-throughput proteomics analysis and mainly for biomarker validation. Similarly, enormous amount of data can be obtained from the cell line macroarray (CLMA) technology, which developed from the TMA using formalin-fixed, paraffin-embedded cell pellets. Here, we applied CLMA technology in stem cell research and in particular to identify bona fide neogenerated human induced pluripotent stem cell (hiPSC) clones suitable for down the line differentiation. All hiPSC protocols generate tens of clones, which need to be tested to determine genetically stable cell lines suitable for differentiation. Screening methods generally rely on fluorescence-activated cell sorting isolation and coverslip cell growth followed by immunofluorescence; these techniques could be cumbersome. Here, we show the application of CLMA to identify neogenerated pluripotent cell colonies and neuronal differentiated cell products. We also propose the use of the automated image analyzer, TissueQuest, as a reliable tool to quickly select the best clones, based upon the level of expression of multiple pluripotent biomarkers.
在过去十年中,组织微阵列(TMA)技术已发展成为一种用于高通量蛋白质组学分析的创新工具,主要用于生物标志物验证。同样,从细胞系宏阵列(CLMA)技术中可以获得大量数据,该技术是从使用福尔马林固定、石蜡包埋的细胞沉淀的TMA发展而来的。在此,我们将CLMA技术应用于干细胞研究,特别是用于鉴定适合后续分化的真正新生成的人诱导多能干细胞(hiPSC)克隆。所有hiPSC方案都会产生数十个克隆,需要对这些克隆进行测试,以确定适合分化的基因稳定细胞系。筛选方法通常依赖于荧光激活细胞分选分离和盖玻片细胞生长,随后进行免疫荧光检测;这些技术可能很繁琐。在此,我们展示了CLMA在鉴定新生成的多能细胞集落和神经元分化细胞产物方面的应用。我们还建议使用自动图像分析仪TissueQuest,作为一种可靠的工具,根据多种多能生物标志物的表达水平快速选择最佳克隆。