Abstract 11015: Development of Serum-Free Suspension Culture System of Peripheral Blood Mononuclear Cells to Potentiate Vascular Regeneration
BACKGROUND: A large amount of peripheral blood (PB) mononuclear cells (MNCs) or PBCD34+ cells following G-CSF administration have been clinically applied to blood cell therapy for vascular regeneration targeting ischemic diseases. To simplify cell source and strengthen therapeutic potential, we tried to develop the methodology of serum-free suspension culture (SFSC) for endothelial progenitor cells (EPCs) using MNCs isolated from low volume PB.
METHODS & RESULTS: SFSC was performed on PBMNCs freshly isolated from healthy human subjects (N= 18), using StemLine II containing SCF, TPO, Flt-3 ligand, VEGF and IL-6 for 7 days. EPC colony forming assay unveiled the augmented frequency of definitive EPC colony forming cells (dEPC-CFCs) by 42 fold in SFSC cells vs PBMNCs, but also the significant expansion of dEPC-CFCs by 19 fold per 100 ml PB, albeit the total cell number decreased. Flow cytometry of SFSC cells vs PBMNCs disclosed the anti-inflammatory macrophage polarization by 2.98 to 0.008 fold in M1/M2 (CCR2/CD206) ratio on the % cell positivity. qPCR of SFSC cells demonstrated the increased expression of angiogenic factor (fold increase = 4.2 or 2.4 for VEGF-B or angiopoietin-1; 35.9, 6.3 or 5.4 for Leptin, IL-8 or IL-10), and inversely the decreased expression of pro-inflammatory factor (fold decrease = 0.23 or 0.44 for IL-1β or TGF- β). Intramuscular cell therapy (Tx) for a hindlimb ischemia model was performed using weight-adjusted cell counts of SFSC cells and PBMNCs (10,000 cells/mouse, corresponding to cell counts gained from 100 ml human PB at most). Laser doppler analysis at day 20 post Tx presented that SFSC cell-Tx significantly recovered the blood flow ratio by 1.8 fold vs PBMNC-Tx, where the ratio was equivalent between PBMNC-Tx and non cell-Tx (N= 12). The histological assessment in SFSC cell-Tx vs PBMNC-Tx, exhibited the increased isolectin B4-FITC+ capillary density by 3.4 fold and the promoted recruitment of SM α actin-Alexa 594+ pericytes by 2.0 fold.
CONCLUSION: SFSC potentiates the vascular regeneration ability of naïve MNCs isolated from extremely low dose PB via the orchestration of promoted effects on EPC expansion, anti-inflammation, angiogenesis and arteriogenesis, thereby providing a practical therapeutic option for ischemic diseases.
- © 2012 by American Heart Association, Inc.