Priming Mononuclear Cells to Improve Outcomes of Regenerative Therapy

I n a seminal report in 1997, Asahara and colleagues 1 first described the existence of endothelial progenitor cells (EPCs) in human peripheral blood (PB) CD34 mononuclear cell (MNC) fraction. In a subsequent report, they described the ability of bone marrow (BM)-derived circulating EPCs to induce neovascularization. The obvious therapeutic potential of EPCs for ischemic diseases has since driven intense research focused on vasculogenic cells from diverse sources with variable phenotypic attributes and biological functions. Although data from clinical trials have shown a modest positive impact of circulating progenitor therapy in cardiovascular and peripheral arterial diseases, new and enhanced methods for cell isolation, expansion, and characterization are critically important to improve regenerative outcomes. Indeed, the rarity of circulating EPCs remains a major hurdle toward successful and wider clinical application using autologous cells. The MNC fraction from PB contains primarily lineage-committed lymphoid and myeloid cells and a very small percentage of CD34 or CD133 stem/progenitor cells. Accordingly, attempts have beenmade tomobilize EPCs into PB with repeated administration of granulocyte colony stimulating factor (G-CSF) followed by apheresis, a common practice in the setting of BM transplantation. Although several clinical trials of tissue regeneration have been completed with progenitors harvested from PB following G-CSF injection, successful expansion of EPCs from PB would be a preferable approach to circumvent the need for G-CSF therapy. Moreover, although BM harvest is a minimally invasive procedure, phlebotomy for PB is less expensive and tolerated better by patients. In this issue of JAHA, Masuda and colleagues report successful enrichment of EPCs from human peripheral blood mononuclear cells (PBMNCs) using a quality and quantity culture (QQc) method and salvage of ischemic limbs in mice with injection of expanded cells. Culture of PBMNCs in QQc medium for 7 days resulted in a 19-fold increase in definitive EPC (dEPC) colony-forming cells, despite a 50% reduction in total number of cells. Moreover, these primed dEPCs showed a 2.7-fold greater endothelial differentiation potential. The frequency of dEPC colony-forming cells correlated positively with the primitive EPC (pEPC) colony-forming cells in PBMNCs, indicating that the QQc method effectively transitioned the pEPC colony-forming cells into dEPC colony-forming cells with increased potential for new vessel formation. Quality and quantity cultured mononuclear cells (QQMNCs) also expressed greater levels of mRNA for angiogenic molecules, including insulin-like growth factor-1 and interleukin (IL)-8, supporting the efficacy of QQc in inducing a vasculogenic phenotype in PBMNCs. Consistent with these favorable alterations in MNC phenotype, the injection of QQMNCs improved limb salvage following hindlimb ischemia in mice. Multimodality assessments showed increased perfusion, angiogenesis, and myogenesis and reduced fibrosis in QQMNC-treated mice. Important from a therapeutic standpoint, compared with G-CSF–mobilized CD34 cell transplantation, QQMNC injection led to equal or greater improvement in outcomes in the setting of hindlimb ischemia. With growing clinical need for EPCs in large numbers, various methods of enrichment and expansion have been developed by different laboratories for EPCs with diverse cellular phenotypes. Consistent with an endothelial or angiogenic theme, the culture medium for this purpose usually contains an endothelial medium (eg, endothelial basal medium-2 and endothelial growth medium-2) with or without serum and angiogenic growth factors. These factors usually include varying combinations and concentrations of vascular endothelial growth factor, fibroblast growth factor-B, insulin-like growth factor-1, epidermal growth factor, hydrocortisone, ascorbic acid, and heparin. Somewhat differently, the QQc medium used in this study by Masuda et al contained Stemline II (Sigma-Aldrich), a hematopoietic stem cell expansion medium, which did not contain any cytokine or The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. From the Division of Cardiovascular Diseases and the Cardiovascular Research Institute, University of Kansas Medical Center, Kansas City, KS. Correspondence to: Buddhadeb Dawn, MD, Division of Cardiovascular Diseases, 3901 Rainbow Blvd, 1001 Eaton Hall, Mail Stop 3006, Kansas City, KS 66160. E-mail: [email protected] J Am Heart Assoc. 2014;3:e001168 doi: 10.1161/JAHA.114.001168. a 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.