Analysis of gene expression in single cells is required to understand somatic cell reprogramming into human induced pluripotent stem cells (iPSCs). To facilitate this, we established intermediately reprogrammed stem cells (iRSCs), pre-iPSC lines. The iRSC-iPSC conversion system enables the reproducible monitoring of reprogramming events and the analysis of progressive gene expression profiles using single-cell microarray analysis and genome editing. Here, single-cell microarray analysis showed the stage-specific sequential gene activation during the conversion of iRSCs into iPSCs, using OCT4, TDGF1 and E-CADHERIN as marker genes. Out of 75 OCT4-related genes, which were significantly up-regulated after the activation of OCT4, and entry into the mesenchymal-to-epithelial transition (MET), LIN28 (LIN28A) and FOXO1 were selected for applying to gene expression visualization. Multicolored visualization was achieved by the genome editing of LIN28 or FOXO1 with mCherry into OCT4-GFP iRSCs. Fluorescent analysis of gene activity in individual cells showed that OCT4 was dispensable for maintenance, but required for activation, of the LIN28 and FOXO1 expression in reprogramming.

中文翻译：

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人中间重编程的干细胞向iPS细胞的顺序转换的可视化。

需要分析单细胞中的基因表达以了解将体细胞重编程为人诱导的多能干细胞（iPSC）。为了促进这一点，我们建立了中间重编程的干细胞（iRSC），iPSC之前的品系。iRSC-iPSC转换系统可使用单细胞微阵列分析和基因组编辑实现可重现的重编程事件监测和渐进基因表达谱分析。在这里，单细胞微阵列分析显示了使用OCT4，TDGF1和E-CADHERIN作为标记基因，在iRSC转换为iPSC的过程中特定阶段的顺序基因激活。在75个与OCT4相关的基因中，这些基因在OCT4激活后显着上调，并进入了间质到上皮的转化过程（MET），选择LIN28（LIN28A）和FOXO1应用于基因表达可视化。通过将带有mCherry的LIN28或FOXO1基因组编辑为OCT4-GFP iRSC，可以实现多色可视化。对单个细胞中基因活性的荧光分析表明，在重新编程时，OCT4对于维持LIN28和FOXO1的表达是必需的，但对于激活是必需的。