Dataset Information

Transcription profiling of mouse inner cell mass (ICM) from a single cells derived from mouse blastocysts at E3.5

ABSTRACT: The inner cell mass (ICM) of the early blastocyst at E3.5, a source of ES cell derivation, is a morphologically homogeneous population of undifferentiated pluripotent cells that give rise to all embryonic lineages. The immediate application of the newly developed V1V3 method to single cells in this stage of mouse embryos revealed the presence of two populations of cells, one with primitive endoderm expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated primitive endoderm and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell, and developmental biology, where small numbers of distinctive or diseased cells play critical roles. Experiment Overall Design: We isolated blastocysts at E3.5 and dissociated the ICM into single cells by trypsin-EDTA treatment. To prepare cDNA samples, we then randomly picked a total of 55 single cells. cDNAs were synthesized and amplified by the V1V3 method, and screened by gene-specific PCR using Oct4 and Cdx2 to remove trophectoderm cells, and 50 cells were identified as Oct4-positive and Cdx2-negative, ICM cells.

OTHER RELATED OMICS DATASETS IN: PRJNA104647PRJNA104667PRJNA94845

INSTRUMENT(S): 418 [Affymetrix]

SUBMITTER: Kazuki Kurimoto

PROVIDER: E-GEOD-4307 | ArrayExpress | 2008-06-13

SECONDARY ACCESSION(S): GSE4307PRJNA104667

REPOSITORIES: GEO, ArrayExpress

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An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.

Kurimoto Kazuki K Yabuta Yukihiro Y Ohinata Yasuhide Y Ono Yukiko Y Uno Kenichiro D KD Yamada Rikuhiro G RG Ueda Hiroki R HR Saitou Mitinori M

Nucleic acids research 20060317 5

A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the repr ...[more]

PMID: 16547197

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Project description:Our computational analysis focuses on the 32- to 64-cell mouse embryo transition, Embryonic day (E3.25), whose study in literature is concentrated mainly on the search for an early onset of the second cell-fate decision, the specification of the inner cell mass (ICM) to primitive endoderm (PE) and epiblast (EPI). We analysed single-cell (sc) microarray transcriptomics data from E3.25 using Hierarchical Optimal k-Means (HOkM) clustering, and identified two groups of ICM cells: a group of cells from embryos with less than 34 cells (E3.25-LNCs), and another group of cells from embryos with more than 33 cells (E3.25-HNCs), corresponding to two developmental stages. Although we found massive underlying heterogeneity in the ICM cells at E3.25-HNC with over 3,800 genes with transcriptomics bifurcation, many of which are PE and EPI markers, we showed that the E3.25-HNCs are neither PE nor EPI. Importantly, analysing the differently expressed genes between the E3.25-LNCs and E3.25-HNCs, we uncovered a non-autonomous mechanism, based on a minimal number of four inner-cell contacts in the ICM, which activates Oct4 in the preimplantation embryo. Oct4 is highly expressed but unstable at E3.25-LNC, and stabilizes at high level at E3.25-HNC, with Bsg highly expressed, and the chromatin remodelling program initialised to establish an early naïve pluripotent state. Our results indicate that the pluripotent state we found to exist in the ICM at E3.25-HNC is the in vivo counterpart of a new, very early pluripotent state. We compared the transcriptomics profile of this in vivo E3.25-HNC pluripotent state, together with the profiles of E3.25-LNC, E3.5 EPI and E4.5 EPI cells, with the profiles of all embryonic stem cells (ESCs) available in the GEO database from the same platform (over 600 microarrays). The shortest distance between the set of inner cells (E3.25, E3.5 and E4.5) and the ESCs is between the E3.25-HNC cells and 2i?+?LIF ESCs; thus, the developmental transition from 33 to 34 cells decreases dramatically the distance with the naïve ground state of the 2i?+?LIF ESCs. We validated the E3.25 events through analysis of scRNA-seq data from early and late 32-cell ICM cells.

			Action	DRS
	E-GEOD-4307.README.txt	Txt
	E-GEOD-4307.eSet.r	Other
	E-GEOD-4307.idf.txt	Idf
	E-GEOD-4307.processed.1.zip	Processed
	E-GEOD-4307.raw.1.zip	Raw