Evidence That Human Very Small Embryonic-Like Stem Cells (VSELs) Are Mobilized by G-CSF Into Peripheral Blood: A Novel Strategy to Obtain Human Pluripotent Stem Cells for Regenerative Medicine.

Abstract 1474 Poster Board I-497 We previously demonstrated that human cord blood contains a population of small (smaller in size than erythrocytes) CXCR4+CD133+CD34+SSEA-4+Oct-4+lin−CD45− cells (Leukemia 2007:21;297-303) and that these cells are mobilized into peripheral blood during tissue organ damage as seen for example in heart infarct (J. Am. Coll. Cardiol., 2009:53;1-9.) or stroke (Stroke. 2009:40;1237.). Similar cells were also reported in murine organs, and more importantly we described that these cells may differentiate in vitro into cells from all three germ layers (Leukemia 2006:20;857–869). To explore the possibility that human VSELs could become a source of pluripotent stem cells in regenerative medicine, our goal was to develop an efficient strategy to isolate these cells from adult patients. To test if VSELs similarly to their murine counterparts could be mobilized into peripheral blood after granulocyte colony stimulating factor (G-CSF) injection (Stem Cells 2008:26;2083-2092), we enrolled a group of young healthy donors who were mobilized for two consecutive days using G-CSF (480 μg/day subcutaneously). On the third day nucleated cells (TNC) were collected by apheresis. We evaluated number of VSELs in peripheral blood (PB) samples before and after G-CSF mobilization as well as the final number in the apheresis product. At least 1 million of TNC were acquired and analyzed by FACS Diva software. Three different fractions of non-hematopoietic stem cells enriched for VSELs (Lin−/CD45−/CD133+, Lin−/CD45−/CD34+, Lin−/CD45−/CXCR4+) as well as their CD45 positive hematopoietic counterparts were analyzed. The absolute numbers of cells from each population, contained in 1 μL of sample, were computed based on percent content of each population and TNC count for each individual sample. Results show that after G-CSF mobilization, human peripheral blood contains a population of lin− CD45− mononuclear cells that express CXCR4, CD34 and CD133 antigens. These lin− CD45− CXCR4+ CD133+ CD34+ cells are highly enriched for mRNA for intra-nuclear pluripotent embryonic transcription factors such as Oct-4, Sox2 and Nanog. More importantly we found that Oct-4 was expressed in nuclei of mobilized VSELs and that these cells also express the cell surface marker SSEA-4, the early embryonic glycolipid antigen commonly used as a marker for undifferentiated pluripotent human embryonic stem cells. We observed that these adult peripheral blood-derived VSELs are slightly larger than their counterparts identified in adult murine bone marrow, but are still very small. In addition, they also possess large nuclei containing embryonic-type unorganized euchromatin. Before G-CSF mobilization only very few VSELs were detectable in peripheral blood, whereas following G-CSF induced mobilization there was a very significant increase with in excess of 106 VSELs present in the apheresis product representing less than 0.01% of TNC. We postulate that while VSELs are relatively rare cells, they are mobilized into peripheral blood and that G-CSF induced mobilization could become a novel strategy to obtain human pluripotent stem cells for regenerative medicine. Disclosures: Medicetty: NeoStem Inc: Employment, Equity Ownership. Marasco: NeoStem Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rodgerson: NeoStem Inc: Employment, Equity Ownership.