Genetic and Epigenetic Studies Reveal That Murine Oct-4⁺ Very Small Embryonic/Epiblast-Like Stem Cells (VSELs) Present in Adult Tissues Share Several Similarities/Markers with Epiblast-Derived Migratory Primordial Germ Cells (PGCs).

Abstract 2521

Poster Board II-498

Recently, we identified a population of pluripotent VSELs in murine adult bone marrow (BM; Leukemia 2006:20;857). Compared with other adult stem cells (SCs), VSELs show unique epigenetic features including: i) open chromatin structures in the promoter of Oct-4 and Nanog; and ii) parent-of-origin-specific reprogramming of genomic imprinting. These features explain the pluripotent embryonic-like nature and quiescent status of these cells, respectively (Leukemia 2009:In press), and indicate their relation to an epiblast/germ-line pluripotent (P)SC population. To better understand the developmental origin of VSELs, we examined gene expression profiles and the epigenetic status of epiblast and germ-line related genes in these cells. We employed real time quantitative PCR (RQ-PCR) to evaluate gene expression, a bisulfite sequencing strategy to evaluate DNA methylation, and chromatin immunoprecipitation (ChIP) to elucidate histone codes in genes of interest. VSELs were isolated from murine BM by multiparameter fluorescence-activated cell sorter (FACS) as a population of Sca⁺lin⁻CD45⁻ along with Sca⁺lin⁻CD45⁺ hematopoietic (H)SCs and BM mononuclear cells (MNCs). We noticed that VSELs, similarly to epiblast PSCs (EpiSCs), highly express the stemness genes (e.g., Oct-4, Nanog, Sox2, Klf4) and epiblast markers (Gbx2, Fgf5, Nodal). However, the Rex1 gene is expressed at a lower level compared to the murine ESC-D3 line. Moreover, VSELs also highly express the Stella, Blimp1, Dnd1, and Nanos3, which are developmental regulators during specification in the proximal epiblast of PGCs. Accordingly, the Stella promoter in VSELs was partially demethylated and highly enriched for transcriptionally active histones (acetylated H3, trimethylated lysine4 of H3) while being simultaneously less enriched for repressive ones (dimethylated lysine9 and trimethylated lysine27 of H3). In particular, we noticed that VSELs resemble migratory PGCs. To support this notion, VSELs: i) express several markers of migratory PGCs (Dppa2, Dppa4, Mvh); ii) do not express post-migratory PGCs genes (Dazl, Sycp3); and iii) have reprogrammed DNA demethylation in the repetitive sequence (LINE1) and the promoters of Mvh, Dazal, and Sycp3. Finally, VSELs express less the transcripts for cMyc, Stat3, Snai, and Ecat1, which are expressed in early specified PGCs. In conclusion, our previous data showing reprogramming of genomic imprinting in VSELs with the present gene expression profile and epigenetic studies strongly supports VSELs developmentally originating from epiblast-derived germ-line SCs (PGCs), particularly migratory PGCs. We believe VSELs are deposited during embryogenesis in the adult tissues as a backup for tissue-committed SCs and that epigenetic reprogramming tightly controls their proliferative potential. Thus, identification of mechanisms that control and modify the epigenetic marks in VSELs will be crucial for developing more powerful strategies to “unleash the power” of these cells and employ them in regenerative medicine.