Umbilical Cord Blood Derived CD133⁺ Cells: Homing Capability in Vasculogenesis and Immunogenicity.

Human CD133⁺ cells constitute a phenotypically and functionally distinct population of endothelial stem and precursor cells that may play a role in postnatal angiogenesis. CD133⁺ homing to stimuli, IL-8 production and immunogenic properties are anticipated important characteristics to consider in potential use of allogeneic UCB CD133⁺ as a therapeutic in arterial ischemia. CD133⁺ cells were isolated from UCB mononuclear cells (MNC) by magnetic bead selection (AutoMACS, Miltenyi Biotech) according to manufacturer’s protocol and analyzed by flow cytometry. Yields per cord blood unit averaged 1.05 x 10⁶ cells (+/− 0.7 SEM n=30). Surface phenotyping of UCB CD133⁺ showed co-expression of VEGFR2 (3.5%), CD105 (22.7%) and CXCR4 (8.7%) ligand for SDF-1. Both HLA-DR (57.6%) and HLA-ABC (66.5%) were expressed on CD133⁺ cells suggesting that CD133⁺ cells may be capable of presenting immunostimulatory antigens and eliciting an allogeneic reaction. To test this, we performed 96 hour mixed lymphocyte reactions (MLR) using healthy adult peripheral blood MNC as responders stimulated by irradiated (30Gy) CD133⁺ from UCB (ratio 3:1). Proliferation was measured by ³H-thymidine incorporation. CD133⁺ and MNC from UCB induced proliferation from allogeneic healthy adult MNC in vitro (46939 +/− 2764 and 49548 +/− 2018 cpm respectively, n=2). MLR studies with CSFE-stained responder cells revealed equivalent rates of lymphocyte cell division comparing selected UCB CD133⁺ and MNC cells used as stimulators. Comparison studies of responding lymphocyte cytokine production including pro-inflammatory protein assays are ongoing. Initial angiogenic protein assays of CD133⁺ cells demonstrated elevated levels of IL-8 production as compared to MNC (10³+/−380 pg/mL greater in CD133⁺ than MNC from the same UCB unit) when cultured for 24h in basal media. Transwell migration assays of CD133⁺ cells to SDF-1 (100ng/mL) demonstrated a 1.8 ± 0.7 fold increase in homing compared to a negative control, coinciding with the CXCR4 expression observed on these cells. In summary, UCB derived CD133⁺ cells demonstrate homing capability as well as potential for cellular recruitment (IL-8 production) for angiogenesis and cellular therapeutics. CD133⁺ cells selected from UCB maintain immunostimulatory capacity and initiate proliferation of adult MNC. Further studies of UCB derived CD133⁺ pro-inflammatory potential; cell recruitment and homing to ischemic signals are warranted.