Format

Send to

Choose Destination
See comment in PubMed Commons below
Dev Biol. 2010 Oct 1;346(1):90-101. doi: 10.1016/j.ydbio.2010.07.021. Epub 2010 Jul 24.

Survival and death of epiblast cells during embryonic stem cell derivation revealed by long-term live-cell imaging with an Oct4 reporter system.

Author information

  • 1Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN-Kobe, Minatojima-minamimachi 2-2-3, Chuo-ku, Kobe City, Hyogo 651-0047, Japan. yamagata@cdb.riken.jp

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.

PMID:
20659443
DOI:
10.1016/j.ydbio.2010.07.021
[PubMed - indexed for MEDLINE]
Free full text
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Support Center