The CD45−LIN− Adult Marrow-Derived CXCR4+ SSEA-1+ Oct-4+ Very Small Embryonic-Like (VSEL) Stem Cells Form In Vitro Spheres Which May Differentiate into CD45+ Hematopoietic Cells.

Abstract

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 (Leukemia 2006; 20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for primary embryonic stem cells such as i) a small size (~2–4 μm in diameter), ii) a large nuclei surrounded by a narrow rim of cytoplasm, and iii) open-type chromatin (euchromatin). These cells isolated freshly from the BM neither grow hematopoietic colonies nor radioprotect lethally irradiated recipients. Recently, however, we noticed that purified VSELs in co-cultures with C2C12 murine sarcoma supportive feeder-layer grow spheres and cells from these VSEL-derived spheres (VSEL-DS) are composed of immature cells with large nuclei containing euchromatin, and similarly as VSELs are CXCR4+SSEA-1+Oct-4+. We found that cells from VSEL-DS after re-plating over C2C12 cells may again (up to 5–7 passages) grow new embryoid-like bodies or if plated into cultures promoting tissue differentiation show pluripotency and expand into cells from all three germ-cell layers. Based on this we tested if CD45− VSEL-DS cells could also differentiate into the hematopoietic lineage. To address this issue we employed similar culture conditions that are employed for hematopoietic differentiation of established embryonic stem cell lines. We noticed that VSEL-DS cells if cultured/passaged in methylocellulose cultures supplemented with hematopoietic growth factors (KL, IL-3, EpO and GM-CSF) give rise to colonies composed of myeloid (CD45+ Gr-1+) and erythroid (Terr-119+) hematopoietic cells. The hematopoietic differentiation of VSEL-DS cells was accompanied by upregulation of mRNA for several genes regulating hematopoiesis (e.g. PU-1, c-myb, LMO2, Ikaros). Based on this we postulate that VSELs that reside in bone marrow may contribute to hematopoiesis. We also provide direct evidence that CD45+ cells may derive from a CD45− population. We postulate that VSELs are closely related to a population of long-term engrafting hematopoietic stem cells and currently we are testing this possibility in animal models.