Dáša Doležalová

Human Embryonic Stem Cells Are Capable of Executing G1/S Checkpoint Activation

Embryonic stem cells progress very rapidly through the cell cycle, allowing limited time for cell cycle regulatory circuits that typically function in somatic cells. Mechanisms that inhibit cell cycle progression upon DNA damage are of particular importance, as their malfunction may contribute to the genetic instability observed in human embryonic stem cells (hESCs). In this study, we exposed undifferentiated hESCs to DNA‐damaging ultraviolet radiation‐C range (UVC) light and examined their progression through the G1/S transition. We show that hESCs irradiated in G1 phase undergo cell cycle arrest before DNA synthesis and exhibit decreased cyclin‐dependent kinase two (CDK2) activity. We also show that the phosphatase Cdc25A, which directly activates CDK2, is downregulated in irradiated hESCs through the action of the checkpoint kinases Chk1 and/or Chk2. Importantly, the classical effector of the p53‐mediated pathway, protein p21, is not a regulator of G1/S progression in hESCs. Taken together, our data demonstrate that cultured undifferentiated hESCs are capable of preventing entry into S‐phase by activating the G1/S checkpoint upon damage to their genetic complement. STEM CELLS 2010;28:1143–1152

Human Embryonic Stem Cells Suffer from Centrosomal Amplification

Propagation of human embryonic stem cells (hESCs) in culture tends to alter karyotype, potentially limiting the prospective use of these cells in patients. The chromosomal instability of some malignancies is considered to be driven, at least in part, by centrosomal overamplification, perturbing balanced chromosome segregation. Here, we report, for the first time, that very high percentage of cultured hESCs has supernumerary centrosomes during mitosis. Supernumerary centrosomes were strictly associated with an undifferentiated hESC state and progressively disappeared on prolonged propagation in culture. Improved attachment to culture substratum and inhibition of CDK2 and Aurora A (key regulators of centrosomal metabolism) diminished the frequency of multicentrosomal mitoses. Thus, both attenuated cell attachment and deregulation of machinery controlling centrosome number contribute to centrosomal overamplification in hESCs. Linking the excessive number of centrosomes in mitoses to the ploidy indicated that both overduplication within a single cell cycle and mitotic failure contributed to generation of numerical centrosomal abnormalities in hESCs. Collectively, our data indicate that supernumerary centrosomes are a significant risk factor for chromosome instability in cultured hESCs and should be evaluated when new culture conditions are being implemented. STEM CELLS 2011;29:46–56