Pluripotent SSEA-1⁺ OCT-4⁺ CXCR4⁺ Sca-1⁺ lin⁻ CD45⁻ Very Small Embryonic Like (VSEL) Stem Cells Reside in Multiple Murine Organs - Are VSEL Epiblast-Derived Functional Pluripotent Precursors for Tissue Committed Stem Cells or Merely Quesiscent Remants from Embryonic Development?.

Several lines of evidence support the hypothesis that pluripotent stem cells (PSC) reside in adult tissues. Recently, we identified in adult bone marrow (BM) a population of very small embryonic like (VSEL) stem cells (

• very small in size (∼3.6 μm),

• CXCR4⁺Oct-4⁺ SSEA-1⁺Sca-1⁺CD45⁻lin⁻,

• respond to SDF-1 gradient and

• possess large nuclei containing unorganized chromatin (euchormatin).

We learned that VSEL in co-cultures with C2C12 cells form embryoid body-like spheres that contain primitive stem cells able to differentiate into cells from all three germ layers (e.g., myocardium, neural tissue and pancreas). In the current studies, we investigated if in addition to BM, VSEL are also present in other murine fetal and adult organs such as liver, heart, brain, kidney, skeletal muscles, pancreas, testes. VSEL were identified and enumerated in adult organs that in order to remove circulating blood were perfused before homogenization and subsequently detected by

• FACS,

• ImageStream analysis,

• microscopic immunofluorescence staining,

• RQ-PCR for expression of pluripotent stem cell markers and finally

• in functional assays in in vitro based on embryoid body-like spheres formation.

We found that murine fetal liver and adult organs contain some rare VSEL-like cells that express several markers expressed by epiblast/germ line stem cells such as SSEA-1, CXCR4, Oct-4, Nanog and MvH. These cells are small, contain unorganized euchromatin and display high telomerase activity. Their size, however may vary depending on the organ from which they are isolated. The number of VSEL-like cells is higher in young animals and decreases with age. We hypothesize that VSEL are direct descendants of the germ lineage which, in order to pass genes on to the next generation, creates soma, and thus is a “mother lineage” for all somatic cell lineages present in the adult body. We envision that VSEL are deposited during embryogenesis in various organs as mother cells for tissue committed stem cells and that they survive in these locations into adulthood. VSEL, however erase their somatic imprint (differential methylation of IGF-2 and H19 on maternal and paternal chromosmes). This mechanism of erasure protects the developing organism from the possibility of teratoma formation. However, at the same time it also affects some of the aspects of the “true pluripotentiality” of these cells (e.g., their ability to complete blastocyst development). We noticed that at least in BM VSEL are a population of PSC that may differentiate along hematopoietic lineage and give rise to hematopoietic stem cells (HSC). Thus VSEL, fulfill criteria of long-term repopulating HSC. Based on this we postulate that, VSEL could play an important role as an organ-residing reserve population of PSC that generates stem cells committed to particular organs and tissues. Decrease in VSEL number that reside in various organs correlates with senescence and exhaustion of the regenerative potential.