Fat Metabolism and Stem Cell Group, Singapore Bioimaging Consortium, A*STAR, 11 Biopolis Way, Singapore, 138667, Singapore.
Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, A*STAR, 11 Biopolis Way, Singapore, 138667, Singapore.
Stem Cell Res Ther. 2021 Feb 5;12(1):113. doi: 10.1186/s13287-021-02171-6.
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.
尽管近年来在诱导多能干细胞（iPS 细胞）的方法开发和生物学理解方面取得了快速进展，但监测人 iPS 细胞早期重编程过程的工具相对较少。
我们筛选了专门结合人 iPS 细胞的内部荧光文库化合物。经过三次筛选，选择的探针用于使用高内涵成像分析在时间依赖性的方式检测重编程细胞。将探针与不同重编程方法、细胞类型和细胞培养条件下的常规染料进行比较。使用荧光探针进行细胞分选，通过 RNA-seq 分析早期重编程细胞的多能特征和全基因组基因表达特征。最后，研究候选重编程因子对调节重编程效率的能力。
我们鉴定出一种新型 BODIPY 衍生的荧光探针 BDL-E5，它可在早期重编程阶段检测活的人 iPS 细胞。BDL-E5 可在 iPS 集落形成前约 7 天识别出真实的重编程细胞，并与传统的多能标志物呈阳性反应。BDL-E5 分选重编程细胞可产生更多数量和更高质量的 iPS 细胞。BDL-E5 阳性与阴性细胞的 RNA 测序分析揭示了基因表达的早期重编程模式，其中包括 CREB1。过表达 CREB1 可显著提高重编程效率，而敲低 CREB1 则降低重编程效率。
总之，BDL-E5 为描绘早期重编程途径和临床应用的人 iPS 细胞的商业生产提供了有价值的工具。
