Dataset Information

Rejuvenation of pluripotent stem cells by frequent activation of Zscan4, Part A

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE15604 | GEO | 2013/06/12

SECONDARY ACCESSION(S): PRJNA141677

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

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)

Project description:The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, due to a poor understanding of the initial reprogramming mechanism, it is difficult to control the efficiency and quality of the iPSCs. Here we show that Zscan4, expressed transiently in 2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Unlike other factors, Zscan4 is required only for the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process through the recapitulation of the early embryonic program. V6.5 ES cells derived from an F1 hybrid strain (C57BL/6 x 129/Sv) were purchased from Thermo Scientific Open Biosystems. ES cells were cultured at 37C in 5% CO2 in a complete ES medium: DMEM, 15% FBS, 1000 U/ml LIF (ESGRO, Chemicon), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM GlutaMAX, 0.1 mM -mercaptoethanol, penicillin, and streptomycin. V6.5 ES cells (5x105 cells) in suspension were cotransfected with 2 g of a linearized pCAG-Zscan4-ERT2 vector and 0.4 g of a PL452 vector (Liu et al., 2003) (a neomycin-resistant gene driven by a PGK promoter) using the Effectene (QIAGEN) according to the manufacturer s protocol, and plated in 100 mm cell culture dishes. After selecting with G418 for 8 days, resulting ES cell colonies were picked, expanded, and frozen. Subsequently, an ES-ZERT cell clone was selected based on the results of genotyping, qPCR, and puromycin-resistance. ES-ZERT cells (10-15 cells) were injected into 2N blastocysts and then transferred to E2.5 recipient females. After genotyping the pups, ZERT chimeric mice carrying pCAG-Zscan4-ERT2 DNA were established.

Dataset Information

Rejuvenation of pluripotent stem cells by frequent activation of Zscan4, Part A

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets