Abstract 1144: Successful Surface-Aminated Nanofiber Expansion of Human Umbilical Cord-Derived CD133+ Cells Leads to Augmentation of Angiogenic Functionality In Vitro and In Vivo
Background: Umbilical cord blood (UCB) and marrow-derived CD133+ cells have been shown to mediate encouraging effects on therapeutic angiogenesis in both animal models and early clinical trials. Low numbers of CD133+ cells derived from a single donor have been a limitation of use of these cells in cardiovascular therapy. We hypothesized that an ex vivo aminated nanofiber system combined with cytokine supplementation would provide optimized topographical and biochemical signals to allow the expansion and potential functional augmentation of CD133+ cells without promoting terminal differentiation.
Methods and Results: Human UCB derived CD133+ progenitor cells were isolated by MACS sorting and ex vivo expanded on aminated nanofiber plates with cytokine rich media. Cells harvested 10 days after expansion demonstrated a 225X increase in total number. Flow cytometric analysis demonstrated CD133–24%, CD34–93%, CXCR4–97%, LFA-97% surface expression. The expanded cells can uptake AcLDL efficiently and demonstrate a 2.3X increase in transwell migration to SDF-1 as compared to fresh UCB CD133+ cells. In vitro analysis revealed that expanded cells have potential to differentiate into endothelial or smooth muscle phenotype as demonstrated with CD31, vWF, VCAM-1 and F-pholloidin, α-actin, and SM myosin heavy chain immunocytochemistry when re-cultured for 14d in EGM2 or SMBM respectively. RT-QPCR analysis of 1% O2 exposed (hypoxic) cells demonstrated a 2X increase in VEGF and 3X increase in IL-8 gene expression compared to normoxic control. In vivo functionality in a NOD/SCID mouse hind limb ischemic model demonstrated that mice treated with 5 x 106 expanded cells (n=7) augmented blood flow ratio (ischemic/control limb) as compared to mice treated with CD133+ cells (n=7) and control (n=7) at 28d. (control 0.32±.02 vs. UCB133+ 0.37±.02 vs. expanded cells 0.50±.04 p<0.01) Capillary density in ischemic hind-limb was increased at 28d (control 62.5±5.4 vs. expanded cell 97.6±2.5 p< 0.01)
Conclusions: These studies demonstrate successful high level expansion of UCB derived CD133+ cells into functionally potent stem cells which have the capacity to differentiate into vascular cells and promote in vivo neovascularization.