RhG-CSF Mobilized and Apheresis-Collected Endothelial Progenitor Cells for Therapeutic Vasculogenesis.

Endothelial progenitor cells (EPCs) have been identified as part of hematopoietic tissue-derived progenitor cells that are mobilized into the peripheral blood (PB) as a result of tissue injury. It therefore seems likely that circulating EPCs have therapeutic potential by aiding in the neovascularization of ischemic tissue. It was the purpose of this study to provide clinical data on the availability of circulating EPCs at steady-state and after rhG-CSF mobilization, and their collection by leukapheresis. Eight healthy donors underwent rhG-CSF (10 μg/kg) treatment over 4 days. Continuous-flow leukapheresis (COBE Spectra, Version 4.7) was performed on the fourth day of rhG-CSF treatment. Blood samples were drawn before starting rhG-CSF treatment, before apheresis, and from the apheresis collection bag. The total cell numbers collected per apheresis were based on processing a median of 17 L [10.7–20.1] blood volume or approximately three times the donor’s total blood volume. Blood and apheresis samples were analyzed by flow cytometry for EPC surface markers CD34, CD133, VEGFR-2, and by forming EPC colonies (CFU-EPC). The Wilcoxon matched-pairs signed-ranks test was used to compare the distributions at various sampling points.

All CD133 subsets were CD14 negative to exclude differentiated monocytes or macrophages. The median steady-state PB concentrations of circulating CD34⁺CD133⁺VEGFR-2⁺ cells was 0.9/μl [0–3], or 1.7/μl [0.9–4] for CD34⁺133⁻VEGFR-2⁺ EPCs. After 4 days of rhG-CSF mobilization treatment the PB CD34⁺CD133⁺VEGFR-2⁺ subset concentration increased by a median of 8-fold [p 0.01], and the CD34⁺133⁻VEGFR-2⁺ subset concentration by a median of 10-fold [p 0.01] over baseline. The median PB concentration of circulating CFU-EPCs increased by 10-fold [p 0.02]. The median absolute number of CD34⁺CD133⁺VEGFR-2⁺ and CD34⁺CD133^-VEGFR-2⁺ cells collected by leukapheresis per kg of body weight was 0.845 x 10⁶ [0.12–2.55] and 1.54 x 10⁶ [0.06–2.4], respectively. A small population of CD133⁺34⁻ VEGFR-2⁺ cells was identified in steady state and mobilized PB, and in the apheresis collect (0.11 x 10⁶/kg [0–0.26]). CD34⁻ progenitor cells expressing CD133 are believed to represent a primitive progenitor cell population containing SCID-repopulating cells. VEGFR-2 coexpression of those immature cells may define a circulating cell population that contributes to vasculogenesis.

Our data suggest that clonogenic circulating EPCs can be mobilized by rhG-CSF and collected by continuous-flow leukapheresis generating large numbers of EPCs, specifically in the range of 60 x 10⁶ EPCs per leukapheresis procedure for a 70 kg individual. Circulating EPCs represent a novel blood cell component that can be clinically used in large quantities, either unmanipulated or EPC-selected, for therapeutic vasculogenesis.