Lyar contributes to cell cycle progression and multi-lineage differentiation in mouse embryonic stem cells

Ly-1 antibody reactive clone gene (Lyar) is involved in the regulation of embryonic stem cell (ESC) self-renewal. To explore the specific role of Lyar in cell cycle progression and embryonic differentiation, we generated Lyar knockout (KO) mouse ESC (mESC) lines using CRISPR/Cas9, and investigated the effects of Lyar deficiency on mESC proliferation, cell cycle, apoptosis and multi-lineage differentiation. We found that Lyar deficiency reduces proliferation, increases apoptosis, and elevates p53 and p21 protein expression. The impaired mESC proliferation is associated with the increased apoptosis and cell cycle progression defect, which is driven by p53-p21 pathway activation. In embryoid body (EB) formation assay, loss of Lyar led to significant downregulation of most germ layer-specific markers in KO mESC clones, including mesoderm (Gsc, T), endoderm (Gata4, Sox17) and ectoderm marker Pax6. These findings confirm that Lyar is required for cell cycle progression, proliferation, and lineage-specific marker expression during early differentiation, demonstrating that Lyar may serve as a critical regulatory factor in stem cell biology.