Cell cycle-dependent calcium oscillations in mouse embryonic stem cells

Abstract

During cell cycle progression, somatic cells exhibit different patterns of intracellular Ca2+signals during the G0phase, the transition from G1to S, and from G2to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap phases G1and G2, we aimed to determine whether mouse ES (mES) cells still exhibit characteristic changes of intracellular Ca2+concentration during cell cycle progression. With confocal imaging of the Ca2+-sensitive dye fluo-4 AM, we identified that undifferentiated mES cells exhibit spontaneous Ca2+oscillations. In control cultures where 50.4% of the cells reside in the S phase of the cell cycle, oscillations appeared in 36% of the cells within a colony. Oscillations were not initiated by Ca2+influx but depended on inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+release and the refilling of intracellular stores by a store-operated Ca2+influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca2+oscillations were confined to the G1/S phase (∼70% oscillating cells vs. G2/M with ∼15% oscillating cells) of the cell cycle. ATP induced Ca2+oscillations, and activation of SOC could be induced in G1/S and G2/M synchronized cells. Intracellular Ca2+stores were not depleted, and all three IP3receptor isoforms were present throughout the cell cycle. Cell cycle analysis after EGTA, BAPTA-AM, 2-aminoethoxydiphenyl borate, thapsigargin, or U-73122 treatment emphasized that IP3-mediated Ca2+release is necessary for cell cycle progression through G1/S. Because the IP3receptor sensitizer thimerosal induced Ca2+oscillations only in G1/S, we propose that changes in IP3receptor sensitivity or basal levels of IP3could be the basis for the G1/S-confined Ca2+oscillations.