Reprogramming Bone Marrow Stem Cells to Functional Endothelial Cells in a Mini Pig Animal Model

BACKGROUND The aims of this study were to compare the morphological, biochemical, and functional properties of reprogrammed bone marrow stem cell (BMSC)-derived arterial endothelial cells (AECs) and venous endothelial cells (VECs), following adenosine triphosphate (ATP)-stimulation in a mini pig animal model. MATERIAL AND METHODS Bone marrow aspiration was performed in six adult mini pigs. Harvested mononuclear cells were isolated, cultured, and treated with vascular endothelial growth factor (VEGF) (16 µg/ml). Transformed cells were characterized using immunofluorescence staining for CD31 and von Willebrandt factor (vWF) and expression of endothelial nitric oxide synthase (eNOS). Cell release of nitric oxide (cNO) was measured using spectrophotometry. Matrigel assays were used to investigate angiogenesis in transformed BMSCs. RESULTS Reprogrammed BMSCs in culture showed a typical cobblestone-like pattern of growth. Immunofluorescence staining was positive for CD31 and vWF expression. Expression of eNOS, using immunofluorescence staining and Western blot, showed no difference between the reprogrammed BMSCs and VECs. Spectrophotometric examination following stimulation with 10mmol/l ATP, showed comparable cNO release for reprogrammed BMSCs (10.87±1.76 pmol/106 cells/min) and VECs (13.23±2.16 pmol/10^6 cells/min), but reduced cNO release for AECS (3.44±0.75 pmol/10^6 cells/min). Matrigel assay for angiogenesis showed vascular tube formation of differentiated BMSC endothelial cells (grade 3.25). BMSCs cultured without VEGF did not demonstrate vascular tube formation. CONCLUSIONS The findings of this study showed that eNOS expression and release of NO could be used to show that BMSCs can be reprogrammed to functional VECs and AECs.