Dataset Information

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment.

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE40495 | GEO | 2016/05/04

SECONDARY ACCESSION(S): PRJNA174400

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:The gold standard for examining pluripotency of stem cells is to see whether cells can contribute to entire body of animals. Here we show that the increased frequency of Zscan4 activation in mouse ES cells not only enhances, but also maintains their developmental potency in long-term cell culture. As the potency increases, even a whole animal can be produced from a single ES cell injected into 4N blastocyst at unusually high success rate. Although Zscan4-activated cells express genes that are also expressed in 2-cell stage mouse embryos, transiently Zscan4-activated state of ES cells is not associated with the high potency of ES cells. It is thus concluded that ES cells acquire higher potency by going through transient Zscan4 activation state more frequently than the regular state. Taken together, our results indicate that frequent activation of Zscan4 can rejuvenate pluripotent stem cells. MC1 ES cells (for pZscan4-Emerald transfection) and R26R6 ES cells (for pZscan4-Cre ERT2 transfection) were grown on gelatin in 6-well plate, 5x10^5cells in suspension were transfected with 1ug of linearized pZscan4-Emerald vector or pZscan4-Cre ERT2 vector using Effectene (QIAGEN) according manufacturer protocol, and plated in gelatin coated 100 mm dishes. Cells were selected with 5 ug/ml blasticidin, colonies were picked on the 8th day, expanded and frozen for further analysis. DNA microarray analysis of pZscan4-Emerald cells was carried out as described. In brief, universal Mouse Reference RNA (Stratagene) were labeled with Cy5-dye, mixed with Cy3-labeled samples, and used for hybridization on the NIA Mouse 44K Microarray v2.2 (manufactured by Agilent Technologies #014117). The intensity of each gene feature was extracted from scanned microarray images using Feature Extraction 9.5.1.1 software (Agilent Technologies). Microarray data analyses were carried out by using an application developed in-house to perform ANOVA and other analyses (NIA Array Analysis software; http://lgsun.grc.nia.nih.gov)

Dataset Information

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure