High Potential Cord Blood Mesenchymal Stem Cells with Angiogenic Properties for Endothelial Regeneration.

Abstract 2588

Mesenchymal stem cells (MC) represent a promising population for regenerative medicine. Recently, their presence has been documented not only in bone marrow, but also in cord blood (CB), an attractive stem cell source with no ethical controversy. In this study, we investigated if CB could represent a new therapeutic tool for the treatment of severe vascular diseases.

We first validated a negative immunoselection approach using a cocktail of antibodies that targets and reduces erythroid components in order to efficiently isolate low-potential (lp)-CBMSC (46% of isolation efficiency) and high-potential (hp)-CBMSC (8.1% of isolation efficiency). Specific aim of this study was to investigate the proangiogenic effect of hp-CBMSC for endothelial regenerative purposes.

Previous experiments had shown that lp-CBMSC are able to grow until passage 4–5 only, with a lower differentiation rate as compared to the hp-CBMSC, therefore we used hp-CBMS. First, they were induced towards the endothelial lineage by growing them in endothelial medium supplemented with 5% FBS, 100 ng/ml Vascular Endothelial Growth Factor (VEGF) and 50 ng/ml Endothelial Growth Factor (EGF) in 24-well cell culture plates coated with fibronectin. After 3 weeks of induction, the ability of induced-cells to self-organize into capillary-like structures in the presence of basement membrane matrix was analyzed. Human umbilical vein endothelial cells (labeled with green fluorescent dye PKH67) were co-cultured with induced (A) or non-induced (B) hp-CBMSC (labeled with red fluorescent dye PKH26) in the presence of hp-CBMSC conditioned media (supernatant collected from hp-CBMSC cell culture). Interestingly, the tube formation was observed only in the presence of induced-hp-CBMSC (A) where the contribution of the endothelial (green) and mesenchymal (red) compartment in the vascular network was extremely clear and evident. In addition, to evaluate whether there was a possible endothelial contribution of the conditioned medium released by the non-induced-hp-CBMSC, an angiokit assay (Angiokit, TCS CellWorks Ltd) was performed. A strong tube formation was observed and the anastomosis-like structure was confirmed by the expression of CD31 and Von Willebrand Factor (DAB staining) in immunocytochemistry. The next step was to identify the factors released in the conditioned medium and in fact several angiogenic factors were found: HB-EGF 13.5 (8.7 – 16.7); HGF 22.4 (16.3 – 39.1); FGF 10.6 (7.0 – 13.5); VEGF 471.3 (369.3 – 590.0) (the results are expressed as median (and range) pg/mL and normalized per 1×10⁶ seeded cells) using a cytokine array (Searchlight Multiplex Immunoassay Kits). The work is still ongoing for the quantification of the endothelial gene expression by real-time PCR for some angiogenic genes including: angiopoietin-2, the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular endothelium-selectin (E-selectin), VCAM1 and VEGF-A in the induced versus the non-induced hp-CBMSC.

In conclusion, our promising results suggest that hp-CBMSC can be considered as endothelial “drug stores” and are extremely promising paracrine effectors that could be exploited in future clinical applications for endothelial tissue regeneration.