RRC ID 11794
Author Yamagata K, Ueda J, Mizutani E, Saitou M, Wakayama T.
Title Survival and death of epiblast cells during embryonic stem cell derivation revealed by long-term live-cell imaging with an Oct4 reporter system.
Journal Dev. Biol.
Abstract Despite the broad literature on embryonic stem cells (ESCs), their derivation process remains enigmatic. This may be because of the lack of experimental systems that can monitor this prolonged cellular process. Here we applied a live-cell imaging technique to monitor the process of ESC derivation over 10 days from morula to outgrowth phase using an Oct4/eGFP reporter system. Our imaging reflects the 'natural' state of ESC derivation, as the ESCs established after the imaging were both competent in chimeric mice formation and germ-line transmission. Using this technique, ESC derivation in conventional conditions was imaged. After the blastocoel was formed, the intensity of Oct4 signals attenuated in the trophoblast cells but was maintained in the inner cell mass (ICM). Thereafter, the Oct4-positive cells scattered and their number decreased along with apoptosis of the other Oct4-nagative cells likely corresponds to trophoblast and hypoblast cells, and then only the surviving Oct4-positive cells proliferated and formed the colony. All embryos without exception passed through this cell death phase. Importantly, the addition of caspase inhibitor Z-VAD-FMK to the medium dramatically suppressed the loss of Oct4-positive cells and also other embryo-derived cells, suggesting that the cell deaths was induced by a caspase-dependent apoptotic pathway. Next we imaged the ESC derivation in 3i medium, which consists of chemical compounds that can suppress differentiation. The most significant difference between the conventional and 3i methods was that there was no obvious cell death in 3i, so that the colony formation was rapid and all of the Oct4-positive cells contributed to the formation of the outgrown colony. These data indicate that the prevention of cell death in epiblast cells is one of the important events for the successful establishment of ESCs. Thus, our imaging technique can advance the understanding of the time-dependent cellular changes during ESC derivation.
Volume 346(1)
Pages 90-101
Published 2010-10-1
DOI 10.1016/j.ydbio.2010.07.021
PII S0012-1606(10)00952-8
PMID 20659443
MeSH Animals Apoptosis* Cell Line Cell Survival Embryonic Stem Cells / cytology* Female Germ Layers / chemistry Germ Layers / physiology* Male Mice Mice, Inbred C57BL Mice, Inbred DBA Mice, Inbred ICR Octamer Transcription Factor-3 / analysis* Octamer Transcription Factor-3 / genetics
IF 3.262
Times Cited 18
Mice TgN(GOFGFP)11Imeg