Despite recent rapid progress in method development and biological understanding of induced pluripotent stem (iPS) cells, there has been a relative shortage of tools that monitor the early reprogramming process into human iPS cells. We screened the in-house built fluorescent library compounds that specifically bind human iPS cells. After tertiary screening, the selected probe was analyzed for its ability to detect reprogramming cells in the time-dependent manner using high-content imaging analysis. The probe was compared with conventional dyes in different reprogramming methods, cell types, and cell culture conditions. Cell sorting was performed with the fluorescent probe to analyze the early reprogramming cells for their pluripotent characteristics and genome-wide gene expression signatures by RNA-seq. Finally, the candidate reprogramming factor identified was investigated for its ability to modulate reprogramming efficiency. We identified a novel BODIPY-derived fluorescent probe, BDL-E5, which detects live human iPS cells at the early reprogramming stage. BDL-E5 can recognize authentic reprogramming cells around 7 days before iPS colonies are formed and stained positive with conventional pluripotent markers. Cell sorting of reprogrammed cells with BDL-E5 allowed generation of an increased number and higher quality of iPS cells. RNA sequencing analysis of BDL-E5-positive versus negative cells revealed early reprogramming patterns of gene expression, which notably included CREB1. Reprogramming efficiency was significantly increased by overexpression of CREB1 and decreased by knockdown of CREB1. Collectively, BDL-E5 offers a valuable tool for delineating the early reprogramming pathway and clinically applicable commercial production of human iPS cells.

中文翻译：

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用于人类诱导的多能干细胞的新型活细胞荧光探针突出了早期重编程人群

尽管最近在方法开发和对诱导多能干细胞（iPS）的生物学了解方面取得了飞速进步，但相对缺乏监测早期重编程为人iPS细胞过程的工具。我们筛选了与人iPS细胞特异性结合的内部构建的荧光文库化合物。三级筛选后，使用高内涵成像分析法分析所选探针以时间依赖性方式检测重编程细胞的能力。在不同的重编程方法，细胞类型和细胞培养条件下，将探针与常规染料进行了比较。用荧光探针进行细胞分选，以通过RNA-seq分析早期重编程细胞的多能特性和全基因组基因表达特征。最后，研究鉴定出的候选重编程因子的调节重编程效率的能力。我们确定了一种新颖的BODIPY衍生的荧光探针BDL-E5，该探针可在重编程早期检测活的人iPS细胞。BDL-E5可以在iPS集落形成并用常规多能标记阳性染色约7天之前识别出真正的重编程细胞。用BDL-E5对重新编程的细胞进行细胞分选可以产生数量增加和质量更高的iPS细胞。BDL-E5阳性和阴性细胞的RNA测序分析显示了基因表达的早期重编程模式，其中特别包括CREB1。CREB1的过表达显着提高了重编程效率，而敲低CREB1则降低了重编程效率。总的来说，