C/EBPδ regulates cell cycle and self-renewal of human limbal stem cells

Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein delta ( C/ EBPd), Bmi1, and Delta Np63a identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/ EBPd and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/ EBPd inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27(Kip1) and p57(Kip2). These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/ EBP delta, but not Delta Np63 alpha, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self- renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/ EBP delta is recruited to the chromatin of positively ( p27(Kip1) and p57(Kip2)) and negatively ( p16(INK4A) and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity.