Analysis Of The Lin- CD45- CD34/CD133+ Cell Population In G-CSF-Mobilized Peripheral Blood By Polychromatic Flow Cytometry and Micro RNA Profiling

There has been controversy over the existence of rare somatic stem cells in mouse and human bone marrow and in human umbilical cord blood that have been described to be pluripotent. These cells have been observed to lack expression of CD45 and blood cell lineage markers (Lin-), to express markers associated with both hematopoietic (CD34, CD133) and pluripotent (Oct4, Nanog) stem cells, to be smaller than a blood lymphocyte, and have been associated with the ability to differentiate into cells and tissues of all three germ layers. They have been given a variety of names, including Very Small Embryonic/Epiblast-Like (VSEL) cells.

To better characterize these cells, we fractionated G-CSF mobilized adult peripheral blood by elutriation, CD34/CD133 immunomagnetic selection (Miltenyi Biotec, Inc., STEMCELL Technologies, Inc.) and fluorescence-activated cell sorting (FACS) using a MoFlo XDP cell sorter (Beckman Coulter, Inc.). Cell fractions were analyzed on a Beckman Coulter Gallios flow cytometer using a 6-color cocktail of antibodies to CD45, CD34, CD133 and blood cell lineage markers, together with membrane-permeable (DRAQ5) and impermeable (7-AAD) nuclear dyes that distinguish live nucleated cells from dead cells, extracellular vesicles and cell debris.

We observe that over 95% of Lin- CD45- flow cytometry events are extracellular vesicles rather than nucleated cells, and have isolated a population of Lin- CD45- CD34+ vesicles from the earliest elutriation fractions (<35 ml/min counterflow). Rare Lin- CD45- live nucleated cells are also clearly evident, many of which express CD34 but not CD133. This population of Lin- CD45- CD34+ CD133- cells makes up approximately 0.003% of the mononuclear cell population in G-CSF mobilized peripheral blood (1 in 300,000 mononuclear cells, or approximately 1 for every 1,000 CD34+ CD45+ hematopoietic stem/progenitor cells). Lin- CD45- CD133+ live nucleated cells are also identified but are considerably more rare (approximately 1 in 10 million mononuclear cells). Similar cell and vesicle populations are also found in umbilical cord blood, although with frequencies about 10-fold higher than in mobilized adult blood.

To begin to characterize these cell and vesicle fractions, we isolated total RNA from FACS-sorted Lin- CD45- CD34/CD133+ (CD34+ and/or CD133+) cells for miRNA expression profiling (Agilent SurePrint G3 Human v16 microRNA 8x60K microarray, representing 1205 Human miRNAs, 1199 verified as real miRNAs in miRbase 18). Array data were processed using a proprietary normalization algorithm (Sistemic, Ltd.) to generate miRNA expression profiles which were analyzed by microRNA-based fingerprinting (SistemQC™). A robust miRNA profile was generated from the initial Lin- CD45- CD34/CD133+ cell sample, with 107 miRNAs reliably detected (a number consistent with other cellular profiles). The detected miRNAs showed a range of expression levels and were expressed well above the limit of detection for the arrays. Further samples will be analyzed to confirm these preliminary findings.

The Lin- CD45- population observed by flow cytometry analysis of human mobilized peripheral blood and umbilical cord blood is heterogeneous, and made up of distinct populations of vesicles and live nucleated cells that variably express CD34 and CD133. The ability to determine miRNA profiles from rare sorted populations such as Lin- CD45- CD34/CD133+ cells will enable the possible further understanding of the function of these cells, as well as the role of miRNAs in regulating their cellular processes. It is also hoped that the data will enhance the understanding of the potential clinical utility of such cells isolated from human blood products.

Jiang:NeoStem, Inc.: Employment. Mallinson:Sistemic, Ltd.: Employment, Equity Ownership. Olijnyk:Sistemic, Ltd.: Employment. Paterson:Sistemic, Ltd.: Employment. Ridha:Sistemic, Ltd.: Employment. Tang:NeoStem, Inc.: Employment. O'Brien:Sistemic, Ltd.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Fong:NeoStem, Inc.: Employment. O'Neill:NeoStem, Inc.: Employment.