Single Cell based genomewide gene expression analysis of murine bone-marrow derived Very Small Embryonic-Like Stem Cells (VSELs)

Recently, we identified a population of Oct4+Sca-1+Lin-CD45- very small embryonic-like stem-cells (VSELs) in adult tissues. Open chromatin structure of pluripotency genes and genomic imprinting-related epigenetic mechanisms maintain pluripotency and quiescence of VSELs, respectively. However, global transcriptome signature of this rare stem-cell population remains elusive. Here, we demonstrate by genomewide gene-expression analysis with a small number of highly purified murine bone-marrow (BM)-derived VSELs, that Oct4+ VSELs i) express a similar, yet nonidentical, transcriptome as embryonic stem-cells (ESCs), ii) up-regulate cell-cycle checkpoint genes, iii) down-regulate genes involved in protein turnover and mitogenic pathways, and iv) highly express Ezh2, a polycomb group protein. Furthermore, as a result of Ezh2 overexpression, VSELs, like ESCs, exhibit bivalently modified nucleosomes (trimethylated H3K27 and H3K4) at promoters of important homeodomain-containing developmental transcription factors, thus preventing their premature transcription. Notably, spontaneous or RNA interference-enforced down-regulation of Ezh2 during VSEL differentiation removes the bivalent-domain (BD) structure, which leads to de-repression of several BD-regulated genes. Therefore, we suggest that VSELs, like other pluripotent stem-cells, maintain their pluripotent state through an Ezh2-dependent BD-mediated epigenetic mechanism. T7-primed cDNA libraries was synthesized using FACS sorted twenty cells. T7-primed libraries were biotin-labeled using the GeneChip 3’ in vitro transcription (IVT) kit (Affymetrix), starting from ‘‘In vitro Transcription to Synthesize Labeled aRNA. Nine Samples (3 VSELs, 3 HSC, and 3 ESC-D3) are analyzed using GeneChip 3’ Mouse Genome 430 2.0 array (Affymetrix), according to the manufacturer’s instructions.