Different Histone Deacetylase Inhibitors Affect Distinct Cellular Targets within the Hierarchy of Hematopoietic Stem / Progenitor Cells.

Abstract Recently several laboratories have examined the in vitro effects of chromatin modifying agents on hematopoiesis. We have previously reported that the sequential addition of a hypomethylating agent, 5-aza-2′-deoxyctidine (5azaD) and a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) to cultures of human cord blood (CB) CD34+ cells containing SCF, thrombopoietin and FLT-3 ligand (cytokines) resulted in a 10-fold expansion of SCID repopulating cells (SRC) (Araki et al. Blood 2004: 104:881a). Recently others have shown that another HDAC inhibitor, valproic acid (VA) resulted in an expansion of CB CD34+ cells and murine hematopoietic stem / progenitor cells (HSPC) (DeFelice et al. Cancer Res. 2005: 65:1505, Beg et al. Cancer Res. 2005: 65:2537). In our current studies we have compared the efficacy of VA, TSA or 5azaD as single agents or in combination to promote the expansion of CB HSPC in vitro. The frequency and fold expansion of colony forming cells (CFC), cobblestone area-forming cells (CAFC) as well as SRC generated from CB CD34+ cells after 9 days of culture were examined. The addition of cytokines alone result in a 1.5-fold expansion of CD34+CD90+ cells. By contrast the addition of cytokines with VA led to a 65-fold expansion of the numbers of CD34+CD90+ cells as compared to a 1.3-fold, 5.6-fold, 4.2-fold or 12.5-fold expansion of CD34+CD90+ cells in cultures receiving cytokines with 5azaD, TSA or 5azaD/VA or 5azaD/TSA respectively. In vitro biological assays (CFC, CAFC) were performed to determine the correlation between CD34+CD90+ cell expansion and function. Cultures receiving cytokines alone or cytokines with VA had the greatest degree of expansion of CFC (14.4 and 18.6-fold respectively). By contrast cultures receiving cytokines alone contained only 70% of the numbers of CAFC as did the primary CB CD34+ cells. Cultures receiving VA or 5azaD/TSA had the greatest degree of expansion of CAFC numbers (9.6-fold and 11.5-fold respectively). The marrow repopulating potential of these various expanded cell populations were then assayed by transplanting them into NOD/SCID mice. CD34+ cells from cultures receiving cytokines alone or cytokines with 5azaD/VA were devoid of human hematopoietic cell chimerism. By contrast, all NOD/SCID mice receiving grafts from cultures treated with cytokines with 5azaD/TSA had evidence of human multilineage hematopoietic engraftment (7.5% ± 3.7%). Cells from cultures treated with cytokines with VA are capable of engraftment in 2 out of 6 mice with a barely detectable level of human cell chimerism (0.11%, 0.14%). We then assessed using western blot analysis whether the chromatin modifying agents might alter HSC function by upregulating HOXB4 protein levels. HOXB4 protein was detectable in cells cultured in the presence of cytokines with VA, cytokines with 5azaD/VA, cytokines with 5azaD/TSA but only cells treated with cytokines with 5azaD/TSA contained readily assayable SRC. These studies suggest that treatment with different chromatin modifying agents are capable of altering the differentiation program of distinct populations of HSPC. Some treatments (VA, 5azaD/VA) primarily affect CFC and CAFC but not SRC. While 5azaD/TSA targets CAFC and SRC but not CFC. In addition, although HOXB4 may participate in HSC self-renewal, additional genes are likely altered following 5azaD/TSA treatment which are required for the maintenance of SRC potential.