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

Abstract

Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein delta (C/EBPdelta), Bmi1, and DeltaNp63alpha identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBPdelta and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBPdelta 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/EBPdelta, but not DeltaNp63alpha, 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/EBPdelta 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.