Gene expression from pericytes

Background: Blood vessel wall adventitial progenitors may yield a renewable source for therapeutic vasculogenesis in limb and myocardial ischemia models. Donor-related liabilities, epigenetic and expressional changes occurring during stem cell isolation and expansion might result in substantial phenotypic modifications, ultimately impacting on therapeutic activity of the cell product. Methods: Consistency of functional performance was assessed in 15 saphenous vein-derived adventitial progenitor cells (SV-APC) lines, of which 10 derived from leftovers of coronary artery bypass grafts (CABG) and 5 from wastes of varicose SV removal from subjects with no evidence of cardiovascular disease (NC). To correlate the expansion culture data with a clinical outcome, 5 SV-APC lines were transplanted (8x105 cells, im) in mice with limb ischemia (n=5 mice per line). Endpoint measurements of blood flow (BF) by laser Doppler flowmetry, capillary and arteriole density were correlated with epigenetic/expressional markers of transplanted cell populations to predict the outcome of reparative processes in vivo. Results: We report successful expansion in 63% of tested lines, which reached the therapeutic target of 30-50 million viable SVP at passage 8 (P8) in ~10 weeks. Antigenic profile was retained during expansion with stable expression of typical pericyte/mesenchymal markers NG2, PDGFR, CD44, CD90 and CD105 (flow cytometry and immunocytochemistry) and comparative cell line analysis indicated no interference of cardiovascular background. Qualitative differences during expansion show conserved viability and motility capacity and low levels of replicative senescence during expansion in culture. Functional capacity in vitro showed large variability of angiocrine secretion (VEGF-A, Ang1) amongst SV-APC but with no difference based on cardiovascular background and improved network formation with endothelial cell structures on Matrigel. In vivo, SV-APC transplantation induced consistent improvement in BF recovery of ischemic limb, reduced muscle fibrosis and reparative neovascularization. Conclusions: Current protocol generates ready-to-use human SVP in a large number of preparations, with no impact of cardiovascular risk factors on cell product functionality and therapeutic activity. 5 samples characterised by three variables: blood flow, arterial density and capillary density