Abstract 466: Valproic Acid Enhances Human Cord Blood CD34+ Cell Differentiation Toward The Endothelial Phenotype
Background. Human umbilical cord blood (UCB) CD34+ cells are bi-potential progenitors that differentiate into hematopoietic and endothelial cells. Epigenetic control of gene expression by histone deacetylase (HDAC) activity is linked to lineage-decisions in uncommitted stem cells. Here we tested whether HDAC inhibitor valproic acid (VPA) modifies the balance between hematopoietic and endothelial commitment of CD34+ cells.
Methods and results. UCB CD34+ cells selected by magnetic sorting were cultured in serum-free medium containing IL-3 and IL-6 (20 ng/ml), Stem Cell Factor and Flt3L (100 ng/ml) cytokines ±VPA (2.5mM) for 7 days. At day 0 CD34+ cells were 84.2±5.2%. After 1 week in culture CD34 expression exhibited a marked decrease and this effect was inhibited by VPA (CD34+ cells; 90.0±0.8% vs. 30.0±2.0%; n=4; P<0.01). In contrast, VPA had a negative effect on CD34+ cell proliferation, as it induced G0/G1 arrest (cells in G0/G1; 93.0±0.9% vs. 74.1±4.3%; n=3; P<0.05) and decreased the stem cell proliferation index (16.2±2.8% vs. 44.0±8.8%; n=3; P<0.05) as well as total cell number. The negative effect of VPA on cell proliferation was associated with upregulation of p16 and p21 genes. VPA enhanced CD34+ cells stemness; it increased Rhodamine123 dye extrusion by CD34+ cells (Rhodamine 123low; 24.6 ± 6.8% vs 4.7 ± 1.1%; n=3; p<0.05) and enhanced CD34 and GATA-2 genes expression. To assess the effects of VPA on CD34+ cells endothelial vs. hematopoietic commitment it was determined the expression of endothelial and hematopoietic markers at day 7. VPA enhanced CD146+ (76.7±5.7 vs 1.4±0.8; n=3; P<0.05) and Ac-LDL-DiI+ (88.3±6.7 vs 59.3±3.2; n=3; P<0.05) cells. In contrast, CD14+ expressing cells were reduced (1.8±2.6% vs 7.7±4.0%; n=4; P<0.05) and CD31+, CD105+, CD144+ and KDR+ cells were unchanged. Finally, CD34+ cells cultured with VPA for 7 days and plated in endothelial differentiation medium for additional 7 days in fibronectin-coated dishes, formed adherent spindle-shaped cells resembling late EPCs outgrowths while control cells remained unattached and round resembling hematopoietic cells.
Conclusions.VPA modulates UCB CD34+ cells stemness and cell cycle distribution; further, it enhances their commitment toward the endothelial phenotype.