Laboratory of Developmental Epigenome, Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.
Genes Cells. 2019 Oct;24(10):667-673. doi: 10.1111/gtc.12716. Epub 2019 Sep 1.
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.
分析单细胞中的基因表达对于理解体细胞重编程为人类诱导多能干细胞（iPSC）至关重要。为了实现这一目标，我们建立了中间重编程的干细胞（iRSC），即前 iPSC 系。iRSC-iPSC 转化系统能够实现重编程事件的可重复监测，并通过单细胞微阵列分析和基因组编辑分析渐进性基因表达谱。在这里，单细胞微阵列分析显示了 iRSCs 向 iPSC 转化过程中特定于阶段的顺序基因激活，使用 OCT4、TDGF1 和 E-CADHERIN 作为标记基因。在 OCT4 激活和进入间质上皮转化（MET）后，上调的 75 个 OCT4 相关基因中，选择 LIN28（LIN28A）和 FOXO1 用于基因表达可视化。通过将 mCherry 基因组编辑到 OCT4-GFP iRSC 中，实现了 LIN28 或 FOXO1 的多色可视化。单个细胞中基因活性的荧光分析表明，OCT4 对于重编程过程中 LIN28 和 FOXO1 表达的维持是可有可无的，但对于其激活是必需的。
