Noninvasive System for Tracking Naïve Induced Pluripotent Stem Cells During Reprogramming

Abstract

Naïve induced pluripotent stem (iPS) cells are useful for drug development, disease model and regenerative medicine. In female cells, X chromosome reactivation (XCR) is closely linked to the acquisition of naïve iPSCs. To visualize the status of X chromosome in living cells and further analyze XCR mechanism, we established a novel noninvasive detection system of XCR. Here we established female mouse embryonic stem cell (mESC) lines whose allele of both X chromosomes carry each of different fluorescent protein genes [Kusabira Orange (KO) and Enhanced green fluorescent protein (EGFP)]. These mESC clones, initially displaying yellow fluorescence owing to two active X chromosomes, generate chimeric cluster of cells showing either orange or green fluorescence (KO⁺ or EGFP⁺), indicating that X chromosome inactivation (XCI) occurs in these mESC clones during differentiation. When the single colored cells were reprogrammed, the iPS colonies displayed yellow (KO⁺/EGFP⁺) fluorescence, indicating XCR upon reprogramming. Our work reveals generated female KO⁺/EGFP⁺ mESC lines can be used for monitoring X chromosome status during differentiation and/or reprogramming. This novel system is also potential for tracking acquisition of pluripotency of iPSCs during reprogramming and screening for factors or culture conditions which promote this event.