Isolation of G-CSF Mobilized Human Mesenchymal Stem Cells by Elutriation.

Abstract 1183

Mesenchymal stem cells (MSCs) are a population of adult stem cells that constitute a promising source for therapeutic applications. MSCs exist in various adult tissues, such as bone marrow and umbilical cord blood. The most common method for MSC isolation relies on plastic adherence, however, this isolation procedure is hampered by the unpredictable influence of other co-cultured adhering cells. Phenotypic characterization of MSCs is usually carried out by FACS analysis of cell surface marker expression, but no unique marker has yet been identified. To circumvent these limitations, using granulocyte-colony stimulating factor (G-CSF) mobilized adult human peripheral blood (PB), we explored the use of Elutra® Cell Separation System (Caridian BCT) to isolate MSC enriched fractions based on their size and density.

A group of 28 donors were studied after mobilization for two consecutive day of G-CSF injection (480μg/day subcutaneously). Total nucleated cells (TNC) were collected by apheresis on the third day. An average of 6-fold increase in MSCs in the post-mobilized sample was observed. At least 1 million TNC in pre- and post-mobilized PB were analyzed by FACS for the presence of MSCs by cell surface staining with antibodies to CD105, CD90, CD73, CD31, CD45 and CD34. Circa 3 × 10¹⁰ TNC were loaded onto the Elutra to fractionate the apheresis product into several distinct cell fractions. Typically, the MSCs were enriched in 2 fractions, and yielded about 5–10% of the initial loaded cell number. Following culturing and passage, cells from these 2 fractions were grown on plastic with low contamination by other cell types. The cultured cells showed spindle-shaped morphology. Cells were further characterized by FACS analysis and shown to stained positive for mesenchymal specific markers CD90 and CD105; and negative for hematopoietic markers of CD45 and CD34. Furthermore, after cells from all fractions were plated for colony forming unit-fibroblast assay (CFU-F), no colony formation were observed from other than the 2 fractions.

In summary, we demonstrate that MSCs are mobilized into PB by G-CSF and can be collected by apheresis. Size-based Elutra fractionation of apheresis product in a closed system could provide a novel strategy for the large-scale isolation and expansion of human MSCs for autologous cell therapy *Dr. Marasco is a paid consultant for NeoStem and an equity holder.