Metabolic Imbalance Triggers Adaptive Remodeling to Accelerate Diploidization in Murine Haploid Embryonic Stem Cells

ABSTRACT Murine haploid embryonic stem cells (haESCs) are ideal tools for functional genetics analyses because of their single‐genome stem cell features. However, self‐diploidization severely restricts their broader application. Although numerous attempts have been made to prevent diploidization, an effective and reliable strategy is lacking. In this study, we performed multiomics comparative analyses between haESCs and their diploidized counterparts (Di‐haESCs), which revealed that metabolic remodeling induced the adaptive evolution of haESCs toward a diploid state. Notably, an overload of intramitochondrial ROS in haESCs impaired mitochondrial bioenergetics, increasing their susceptibility to cell death and driving the progressive accumulation of diploidized cells in culture. We further found that a disrupted pyruvate–lactate balance in haESCs led to altered tricarboxylic acid (TCA) cycle activity, which was closely linked to mitochondrial dysfunction and haploid instability. Leveraging the recovery of mitochondrial function and a doubled mitochondrial number after diploidization, we performed a genome‐wide screening to identify key mitochondrial quality control (MQC) genes involved in this process. On the basis of these mechanistic insights, we developed a metabolically optimized medium for haploidy maintenance. These findings benefit haploid stem cell‐based genetic screening analyses and deepen the understanding of MQC in mammalian cells.