Expression data from single cells from ICMs of mouse blastocysts at E3.5

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. Keywords: Single cell analysis 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.