Cell cycle-dependent calcium oscillations in mouse embryonic stem cells

Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois Submitted 12 April 2006 ; accepted in final form 4 November 2006 During cell cycle progression, somatic cells exhibit different patterns of intracellular Ca 2+ signals during the G 0 phase, the transition from G 1 to S, and from G 2 to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap phases G 1 and G 2 , we aimed to determine whether mouse ES (mES) cells still exhibit characteristic changes of intracellular Ca 2+ concentration during cell cycle progression. With confocal imaging of the Ca 2+ -sensitive dye fluo-4 AM, we identified that undifferentiated mES cells exhibit spontaneous Ca 2+ 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 Ca 2+ influx but depended on inositol 1,4,5-trisphosphate (IP 3 )-mediated Ca 2+ release and the refilling of intracellular stores by a store-operated Ca 2+ influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca 2+ oscillations were confined to the G 1 /S phase ( 70% oscillating cells vs. G 2 /M with 15% oscillating cells) of the cell cycle. ATP induced Ca 2+ oscillations, and activation of SOC could be induced in G 1 /S and G 2 /M synchronized cells. Intracellular Ca 2+ stores were not depleted, and all three IP 3 receptor isoforms were present throughout the cell cycle. Cell cycle analysis after EGTA, BAPTA-AM, 2-aminoethoxydiphenyl borate, thapsigargin, or U-73122 treatment emphasized that IP 3 -mediated Ca 2+ release is necessary for cell cycle progression through G 1 /S. Because the IP 3 receptor sensitizer thimerosal induced Ca 2+ oscillations only in G 1 /S, we propose that changes in IP 3 receptor sensitivity or basal levels of IP 3 could be the basis for the G 1 /S-confined Ca 2+ oscillations. pluripotent; IP 3 ; store operated Ca entry; IP 3 receptor Address for reprint requests and other correspondence: K. Banach, Dept. of Physiology, Stritch School of Medicine, Loyola Univ. Chicago, 2160 South First Ave., Maywood, IL 60153 (e-mail: kbanac1{at}lumc.edu )