Sriram, S., Kang, N.-Y., Subramanian, S., Nandi, T., Sudhagar, S., Xing, Q., … Sugii, S. (2021). Novel live cell fluorescent probe for human-induced pluripotent stem cells highlights early reprogramming population. Stem Cell Research & Therapy, 12(1). 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.
Attribution 4.0 International (CC BY 4.0)
Development Programme Grant by A*STAR’s Joint Council Office (#1334 k00083)
Please find the link to the article at the publisher's URL: http://dx.doi.org/10.1186/s13287-021-02171-6