BioProject

Pluripotent mouse ES cells maintain their genomic stability via regular passage through the 2-cell-like state (2CLS) with a distinct pattern of gene expression. More...

Pluripotent mouse ES cells maintain their genomic stability via regular passage through the 2-cell-like state (2CLS) with a distinct pattern of gene expression. In a standard culture, only ~5% cells are in the 2CLS; thus, it is desirable to find conditions that facilitate cell transition through this state as a method for improving the quality of ES cells. Here we show that low concentration of retinoic acid (RA) in the presence of LIF increases the proportion of cells undergoing the 2CLS without causing differentiation. Colonies of undifferentiated cells with high contribution of 2CLS marker, Zscan4c, appear clearly by the day 7. These colonies consist of a mixture of three types of cells: (1) cells in the 2CLS, (2) regular pluripotent ES cells that express Pou5f1, Sox2, and Nanog, and (3) endodermal cells, which are located at the periphery of the colony. Cell tracking with LacZ-staining shows that descendants of 2CLS-cells represent the majority of cells in these colonies. Cells exposed to RA and then transferred to the regular medium retain pluripotency, judged by their high ALP staining, typical ES colony morphology, gene expression profile, and stable karyotype. These cells readily differentiate into derivatives of all 3 germ layers in embryoid bodies assay and yield a higher contribution to chimeras after the injection into blastocysts than regular ES cells. In conclusion, transient culture of ES in low concentration of RA could increase the number of ES passaging through 2CLS and thus improve ES pluripotent phenotype and performance in chimera experiments. Overall design: MC1-ZE-3 control day3, MC1-ZE-3 atRA50 48h, and MC1-ZE-3 LY294002 48h are MC1 ES cells cultured in standard conditions. They were treated with 50nM retinoic acid (RA) or with PI3K inhibitor LY294002. RNA was extracted with Trizol (TM) (Invitrogen) 48 h after treatment. MC1-ZE3 day3 cells are cultured in standard conditions, RNA collected on day 3. MC1-ZE16, RA50 day4 cells are treated with 50nM retinoic acid (RA), colonies of pluripotent cells were picked manually on day 4 ES MC1 Intact Lif+ RA cells are treated with 50nM retinoic acid (RA), colonies of pluripotent cells were picked manually on day 7. MC1-ZE3 day3 2nd replication P10 day3 cells were initially treated with RA for 7 days (as in ES MC1 Intact Lif+ RA),and then cultured for 10 passages in standard conditions (without RA). RNA collected on day 3. Less...