Histone Deacetylase Inhibitors Promote the Ex Vivo Expansion of Cord Blood CD34+ Cells in Serum Free Cultures Accompanied by the Upregulation of Pluripotency Genes

Abstract 345

Previously, we have reported that various chromatin modifying agents(CMA) can epigenetically alter HSC fate decisions resulting in increased numbers of cord blood(CB)- SCID repopulating cells(SRC) and/or erythroid progenitor cells depending upon the CMA utilized (Blood. 2011,117(17):4632–41). We have hypothesized that treatment of CB CD34+ cells with histone deacetyalse inhibitors(HDACIs) might expand the numbers of CB-SRC and that such cell products could be used as allogeneic stem cell grafts. We developed serum free(SF) and containing(SC) culture conditions and assessed the ability of several HDACIs including valproic acid(VPA), scriptaid(SCR) and CAY10433(C433) to expand CB-SRC Each of these HDACIs were shown to inhibit both class I and II HDACs. The addition of VPA to SF cultures led to a >6,000 fold expansion of CD34+ cells and >25,000-fold expansion of CD34+CD90+ cells as compared to primary cells(PC). By contrast, SC cultures resulted in only a 54-fold expansion of CD34+ cells and a 75-fold expansion of CD34+90+ cells as compared to PC in the presence of VPA.In order to examine the functional characteristics of HDACIs treated CB-CD34+ cells in SF cultures we evaluated aldehyde dehydrogenase activity(ALDH) in these expanded CD34+ cells. A far greater proportion of CD34+ cells were ALDH+ following treatment with VPA in SF cultures as compared to SC cultures (82.2±8.6%, versus 37.0±0.9%). By contrast incubation in the SC cultures induced the generation of greater proportion of ALDH-CD34+ cells as compared to SF cultures (27.0±5.0% versus 3.7±2.0%). These findings indicate that the inclusion of serum to such cultures favors HSC differentiation and eventual HSC depletion while SF conditions favor expansion of more primitive HPC. Purified ALDH+CD34+ cells from SF cultures supplemented with HDACIs contained far greater absolute numbers of BFU-E and CFU-Mix (8.4×10⁷±6.7×10⁷/CB collection) as compared to SC cultures (1.4×10⁷±8.8×10⁶/CB collection; One way ANOVA p=.02). These data indicate that based upon their degree of ALDH activity and functional potential different phenotypically and functionally defined subsets of CD34+ cells are generated in SF as compared to SC cultures.

In order to further examine the effects of VPA on the pluripotency of expanded CD34+ cells we examined the expression of Sox2, Oct4 and Nanog in CD34+ cells re-isolated following 7 days of treatment with HDACIs in SF and SC cultures. RNA and protein expression levels of Sox2, Oct4 and Nanog genes were greater in cells generated in SF cultures supplemented with HDACIs than that observed in CD34+ cells cultured in SC. Sox2 and Oct4 bind to the promoter of Nanog and up-regulate Nanog expression. In the SF but not SC cultures VPA treatment resulted in the up-regulation of Nanog indicating the functionality of the Sox2/Oct4 interactions. We also documented using confocal microscopy the elevated expression of the proteins derived from these pluripotency genes both in the cytoplasm and nucleus of CD34+ cells re-isolated after a week of treatment with each of the HDACIs.

The functional potential of the re-isolated CD34+ cells following a week of treatment with VPA in SF medium was evaluated by transplanting the expanded cells into NOD/SCID/gamma(c)null mice. SRC capable of multi-lineage hematopoietic differentiation were detected in the marrow of recipients of VPA treated grafts 13-weeks after transplantation (%CD45, 33.5±13.4% with VPA treated grafts versus 13.0±7.7% with grafts generated in the presence of CA). Bone marrow cells from primary recipients receiving HDACI treated CD34+ cells grafts were also capable of generating greater numbers of cells belonging to myeloid and lymphoid lineages(%CD45, 3.3±1.0%) as compared to grafts receiving CA(%CD45, 0.65±0.16%) 15 weeks after their transplantation into secondary recipients.

These data indicate that inhibitors of class I and II HDACs under SF culture conditions provide an effective pharmacological approach which can be utilized to promote the ex vivo expansion of functional CB-SRC which are characterized by high ALDH activity and expression of a variety of pluripotency genes. These findings indicate the importance of epigenetic events in determining phenotype and function of dividing SRC in vitro.