Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA.
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
Stem Cell Reports. 2017 Apr 11;8(4):1101-1111. doi: 10.1016/j.stemcr.2017.03.011.
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost.
将体细胞重编程为诱导多能干细胞 (iPSC) 为研究在活体患者中难以提取的细胞类型(如神经元和心肌细胞)中的人类疾病的分子机制提供了可能。迄今为止,已经发表了一些研究使用小面板的 iPSC 衍生细胞系来研究单基因疾病。然而,为了研究复杂疾病,其中疾病背后的遗传变异尚不清楚,需要生成大量的患者特异性 iPSC 系和对照。目前,衍生和表征 iPSC 的方法既耗时、昂贵,而且在某些情况下,只是描述性的,而不是定量的。在这里,我们着手开发一套简单的方法,以降低成本并提高 iPSC 系表征的通量。具体来说,我们概述了用于高通量量化表面标志物、体外分化潜力的基因表达分析以及具有显著降低成本的染色体组型评估的方法。