A fluorescent chemical probe CDy9 selectively stains and enables the isolation of live naïve mouse embryonic stem cells

Abstract Human and mouse embryonic stem cells (ESCs) differ in terms of their pluripotency status, i.e., naive vs. primed. This affects various biological properties and leads to several technical hurdles for future clinical applications, such as difficulties in chimera formation, single-cell passaging, and gene editing. In terms of generating functional human tissues and organs via mammalian interspecies chimerism, a fluorescent chemical probe that specifically labels naive ESCs would help to isolate these cells and monitor their conversion. This study demonstrates that the fluorescent chemical probe compound of designation yellow 9 (CDy9) selectively stains naive, but not primed, mouse ESCs (mESCs). CDy9 entered cells via Slc13a5, a highly expressed membrane transporter in naive mESCs. Fluorescence-based cell sorting based on CDy9 staining successfully separated naive mESCs from primed mESCs. Mice generated using CDy9 + cells isolated during the conversion of mouse epiblast stem cells into naive mESCs exhibited coat color chimerism. Furthermore, CDy9 specifically stained cells in the inner cell mass of mouse embryos. These findings suggest that CDy9 is a useful tool to isolate functional naive mESCs.