A Lin-CD45-CD34+ Population of Extracellular Vesicles in Human Blood That Mimics Very Small Embryonic-Like Stem Cells (VSELs) by Flow Cytometry
Authors: O'Neill David W. , Jiang Yajuan , Leary Elizabeth , Yavanian Gregory , Eminli Sarah , Marasco Wayne A 
Published: October 2019

Abstract
Abstract 4750
In mice, Very Small Embryonic-like Stem cells (VSELs) are a population of small (4 to 8 μm) Lin-CD45-Sca1+ bone marrow cells that express CD133, CD34 or CXCR4, as well as markers characteristic of embryonic stem cells such as Oct-4 and Nanog. Molecular as well as in vitro and in vivo functional studies indicate that murine VSELs are pluripotent and capable of regenerating damaged host tissues. Although there is some controversy as to whether the Lin-CD45- population in humans contains cells with stem activity, a sub-population of Lin-CD45- cells with a phenotype very similar to murine VSELs has been identified in human umbilical cord blood as well as in adult human bone marrow and G-CSF mobilized peripheral blood (PB); the latter has shown multi-lineage reconstitution in a mouse bone regeneration model. To isolate these human VSELs for further characterization from G-CSF mobilized adult PB, we used a 4-color panel of antibodies (anti-CD45 Pacific Blue, CD34-PE, CD133-PE, FITC-conjugated antibodies to WBC, RBC and PLT lineage markers, and the cell viability dye 7-AAD) to track the cells during purification. We observed that events with the VSEL phenotype (Live Lin-CD45-CD34/133+ flow cytometry events) are relatively rare, approximately 1 for every 40 CD45midCD34+ hematopoietic progenitor cells. However, using methods that separate cells based on size or density such as differential centrifugation, percoll gradient centrifugation, and counterflow centrifugal elutriation, we observed that these Lin-CD45-CD34/133+ events fall into two separate populations with different physical characteristics – a major population (approximately 98% of Lin-CD45-CD34/133+ events) of objects that are very small (< 4 μm), very light, and that stain negatively or dimly with the nuclear dye DRAQ5, and a minor population that is larger (5–10 μm), heavier, and that stains brightly with DRAQ5. FACS sorting of the two populations followed by cytospin and diff-quick stain showed that the minor population consists of small nucleated cells, whereas the major population consists of membrane-bound objects that do not have a cell nucleus. By light microscopy and transmission electron microscopy these objects have the appearance of extracellular vesicles ranging in size from 0.5 to 3 μm. Although roughly the size of platelets, their morphologic appearance is quite different from platelets. The vesicles contain RNA, which should prove useful in determining the origin and possible function of the vesicles. Initial experiments indicate that the vesicles do not stain for CD31, which would suggest they are not of endothelial origin. The two populations of Lin-CD45-CD34/133+ events are also found in umbilical cord blood, although at different frequencies than in mobilized adult blood (1 for every 5 hematopoietic progenitors, with 94% of the events being DRAQ5-). The data indicate that the addition of a nuclear marker such as DRAQ5 is needed to quantify and track true cells having the VSEL phenotype by flow cytometry.

Disclosures:
O'Neill: NeoStem, Inc.: Employment, Stock Options Other. Jiang:NeoStem, Inc.: Employment, Stock Options Other. Leary:NeoStem, Inc.: Employment, Stock Options Other. Yavanian:NeoStem, Inc.: Employment, Stock Options Other. Eminli:NeoStem, Inc.: Employment, Stock Options Other. Marasco:NeoStem, Inc.: Equity Ownership.