Histone Deacetylase Inhibition Enhances Self Renewal and Cardioprotection by Human Cord Blood-Derived CD34+ Cells
Figure 4
Phenotype and stem cell function in control and 7-days VPA treated cells by flow cytometry.
(A) quantification of cells actively extruding Rhodamine123 dye as a result of ABCG2 gene product MDR-1, a typical activity of immature stem cells. Contour plots on the left show the staining profile in the presence of the MDR-1 pump inhibitor Verapamil, plus the CD34 isotype (ISO), while those on the right show the shift toward the left of a CD34bright cells fraction (blue area) in VPA-treated cells (V). Bar graph on the right indicates quantification of CD34bright/Rho123lo cells in C and VPA conditions; dotted line indicate the percentage of CD34bright/Rho123lo cells immediately after isolating CD34+ cells from cord blood. (B) Quantification of ALDH expressing cells. Contour plots on the left show the fluorescence profile of cells treated with the ALDH inhibitor DEAB (used as a negative control) and CD34 isotype antibody (i+D), while those on the right show the results of specific staining with CD34 antibody and fluorescent detection of ALDH activity. Note that in the presence of VPA a higher percentage of CD34bright/ALDH+ cells was present (blue area), while in control cells ALDH staining was lower in the CD34bright gating and present in CD34dim/neg cells (areas in magenta color). Bar graph on the right indicates quantification of CD34bright/ALDH+ cells in C and VPA conditions; dotted line indicate the percentage of these cells immediately after isolating CD34+ cells from cord blood. * indicate P<0.05 by paired t-test (n = 4). (C) phenotype analysis of control and VPA cells at 7 days of culture by multiparametric flow cytometry experiments. Upregulation of stem cells markers CD34, CD133, CD38 and KDR were found along with enhanced expression of mesenchymal markers CD90, CD146 and CD130. Consistent with an effect of VPA on myeloid differentiation inhibition, CD14 was inhibited. * indicate P<0.05 by paired t-test (n≥3). (D) Derivation of ECFCs from fresh, CTR and VPA CD34+ cells. Formation of clusters was observed three weeks after plating these cells onto FN coated dishes. Histogram represents number of ECFC clusters observed in three independent experiments; * indicates P<0.05 by one way ANOVA with Neuman Keuls post-hoc. Pictures on the upper right show the morphology of ECFCs derived from CTR and VPA CD34+ cells and their ability to form capillary-like structures, when plated onto matrigel. The amount of these latter structures formed by either CTR or VPA cells was not different compared with HUVEC cells that were used as a positive control (not shown). Plots in the lower part of the panel show expression of typical ECFC markers [19].