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Recently, we purified a population of CXCR41 /Oct-4 1 /SSEA-1 1 /Sca-1 1 /Lin 2 /CD45 2 very small embryonic-like stem cells (VSELs) from adult murine bone marrow (BM). After using flow cytometry, ImageStream analysis, confocal microscopy, and real time RT-PCR, we report that similar cells could be also identified and isolated from several organs in adult mice. The highest total numbers of Oct-4 1 VSELs were found in the brain, kidneys, muscles, pancreas, and BM. These observations support our hypothesis that a population of very primitive cells expressing germ line/epiblast markers (Oct-4, SSEA-1) is deposited early during embryogenesis in various organs and survives into adulthood. Further studies are needed to determine whether these cells, after being isolated from various adult human organs similarly to their murine BM-derived counterparts, are endowed with pluripotent stem cell properties. ' 2008 International Society for Advancement of Cytometry Key termsVSELs; Oct-4; CXCR4; pluripotent stem cells; ImageStream ADULT bone marrow (BM) tissue has been considered a ''home'' of various types of primitive cells for many years (1,2). As a result of these studies, several types of nonhematopoietic stem cells (HSCs) have been described in adult BM, including: (i) endothelial progenitor cells (EPCs) (3,4); (ii) mesenchymal (M)SCs (5,6); (iii) multipotent adult (MA)PCs (7); and (iv) marrow-isolated adult multilineage inducible (MIAMI) cells (8) as well as precursors of germ cells (oocytes and spermatogonial cells) (9,10). The presence of multiple populations of SCs in the BM could potentially be a result of the ''developmental migration'' of SCs during ontogenesis as well as the presence of the permissive environment that attracts these cells to the BM.Recently, our group purified a population of developmentally primitive SCs from BM, which we named very small embryonic-like SCs (VSELs) (11). These rare cells were isolated by multiparameter fluorescent-activated cell sorting (FACS) as a population of Sca-12 cells. Using transmission electron microscopy (TEM), we observed that these VSELs contain relatively large nuclei filled with a primitive type of chromatin (11). Further analysis using the ImageStream system (ISS) confirmed the particularly small size (\5 lm) of BM-derived VSELs as well as a high nuclear-to-cytoplasmic (N/C) ratio, thus indicating their primitive characteristics (12). We have also found that VSELs are enriched in multiple tissue-specific markers, including early skeletal muscle, heart muscle, neural, liver, intestinal epithelium, and skin epidermis as well as endocrine pancreas markers. We confirmed their pluripotency by demonstrating their ability to differentiate into cellular lineages from all three germ layers in vitro (11,13,14 MATERIALS AND METHODS AnimalsThese experiments were performed in accordance with the guidelines set forth by the University of Louisville's Institutional Animal Care and Use Committee (IACUC). The investigation conforms to the guide for the Care and Use of L...

Pluripotent very small embryonic/epiblast derived stem cells (VSELs) as we hypothesize are deposited at begin of gastrulation in developing tissues and play an important role as backup population of pluripotent stem cells (PSCs) for tissue committed stem cells (TCSCs). We envision that during steady state conditions these cells may be involved in tissue rejuvenation and in processes of regeneration/repair after organ injuries. Molecular analysis of adult bone marrow (BM)-derived purified VSELs revealed that they i) express pluripotent stem cells markers e.g., Oct4, Nanog, Klf-4, SSEA-1 ii) share several markers characteristic for epiblast as well as migratory primordial germ cells (PGCs), and iii) possess a unique pattern of genomic imprinting (e.g., erasure of differently methylated regions at Igf2-H19 and Rasgrf1 loci and hypermethylation at KCNQ1 and Igf2R loci). This supports that VSELs are related to epiblast-derived migrating PGC-like cells and, despite their pluripotent stem cell character, changes in the epigenetic signature of imprinted genes keep these cells quiescent in adult tissues and prevent them from teratoma formation. In contrast epigenetic changes/mutations that lead to activation of imprinted genes could potentially lead to tumor formation by these cells. Mounting evidence accumulates that perturbation of expression of imprinted genes is a common phenomenon observed in developing tumors.