An Evidence That Murine Marrow-Derived CXCR4⁺ SSEA-1⁺ Oct-4⁺ Very Small Embryonic-Like (VSEL) Stem Cells Are Pluripotent and Express Several Primordial Germ Cell (PGC) Markers - Hypotesis for Developmental Deposition of PGC in Various Organs.

Recently we identified in murine BM a homogenous population of rare (~0.01% of BMMNC) Sca-1⁺ lin⁻ CD45⁻ cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (

1. i) small size (2–4 um in diameter),

2. ii) large nuclei surrounded by a narrow rim of cytoplasm, and

3. iii) open-type chromatin (euchromatin).

We also found that VSELs may be released from BM and circulate in peripheral blood during tissue/organ injuries (e.g., heart infarct, stroke). Recently we noticed that ~5–10% of purified VSELs if plated over a C2C12 murine sarcoma cell feeder layer are able to form spheres that resemble embryoid bodies. Cells from these VSEL-derived spheres (VSEL-DS) are composed of immature cells with large nuclei containing euchromatin, and similarly as purified VSELs are CXCR4⁺SSEA-1⁺Oct-4⁺. Furthermore, VSEL-DS after replating over C2C12 cells may again (up to 5–7 passages) grow new spheres or if plated into cultures promoting tissue differentiation expand into cells from all three germ-cell layers. The formation of VSEL-DS was observed in a presence of C2C12 cells obtained from different sources. Furthermore, VSELs isolated from GFP+ mice grew GFP+ VSEL-DS which show a diploid content of DNA. This suggests that VSEL-DS are in fact derived from VSELs and not from the supportive C2C12 cell line as well as excludes the possibility of cell fusion to the observed phenomenon. Similar spheres were also formed by VSELs isolated from murine fetal liver, spleen and thymus. Interestingly formation of VSEL-DS was associated with a young age, and no VSEL-DS were observed by cells isolated from old mice (> 2 years). We also found that cells isolated from VSEL-DS similarly as embryonic stem cells grow tumors after injection into immunodeficient NOD/SCID mice (51/52 inoculated mice). Since VSELs isolated by us express several markers of primordial germ cells (fetal-type alkaline phosphatase, Oct-4, SSEA-1, CXCR4, Mvh, Stella, Fragilis, Nobox, Hdac6) we hypothesize that VSELs are closely related to a population of primordial germ cells. These cells are specified during early gastrulation in the proximal epiblast and subsequently migrate in a CXCR4-SDF-1 dependent manner through the embryo proper to their final destination in genital ridges. It is possible that some of these cells or a population of cells closely related to them migrate astray being chemoattracted by SDF-1 to fetal liver and subsequently, during the third trimester of gestation seed together with hematopoietic stem cells in bone marrow and perhaps other organs as well. In conclusion, we postulate that VSELs identified by us and purified at the single cell level could become an important source of pluripotent stem cells for regeneration.