In Vivo Treatment with Chromatin Modifying Agents Dramatically Increases Hematopoietic Stem Cell Numbers.

Abstract 370

The effects of aging in hematopoietic stem cells (HSC) are still unclear. The experimental evidence suggests a progressive decline in immune function and a possible qualitative or quantitative defect of HSC with aging resulting in ineffective production of functional blood cells in some individuals. This is evidenced by the increased incidence of diseases (i.e. aplastic anemia, hypoplastic myelodysplastic syndrome) characterized by a failure to produce optimal numbers of functional blood cells with advancing age. In order to directly assess functional potency of HSC with aging, HSC from young (27 yo) and elderly (68 yo) healthy human subjects were injected simultaneously into humanized mice. Our results demonstrated that an equal dose of BM CD34+ cells (3.75 × 10⁴) from young individuals always out competed grafts from elderly subjects in a true competitive repopulation assay. We have previously demonstrated that ex vivo treatment of cord blood (CB) CD34+ cells using a hypomethylating drug, 5aza-2-deoxycytidine (5azaD), and an HDAC inhibitor, trichostatin A (TSA), augments the number of repopulating HSC (Araki et al, Blood 2007). We hypothesized whether administration of chromatin modifying agents (5azaD/TSA) in vivo in non-toxic doses could similarly reactivate genes critical for expansion of residual HSCs of a host. To test this hypothesis we have examined whether administration of this therapy (5azaD/TSA) augments human hematopoietic engraftment established from a low number of transplanted HSC in a xenotransplantation assay (NOD/SCID mice). Our results indicate that sequential administration of 5azaD/TSA in vivo 4 to 5 weeks after transplantation can boost the degree of human HSC engraftment. Briefly, when we injected 2 × 10⁴ CD34+CD90+ CB cells and examined the BM of NOD/SCID mice 8-12 weeks after transplantation, the human hematopoietic engraftment was 5.37% in control (n = 11) and 9.26% (n = 17) in mice receiving 5azaD/TSA as measured by flow cytometry (human CD45+, CD33+, CD71+ cells). Similarly when 4 × 10⁴ CD34+CD90+ CB cells were injected the degree of hematopoietic engraftment in 5azaD/TSA treated mice was significantly greater than in the control group (n = 5 each group, 4.03% vs. 27.99%, p = 0.02). In additional experiments, in which the NK cell function of the host was blocked by TM-bf° antibody, the degree of multilinage hematoipoietic chimerism was more pronounced in mice treated with 5azaD/TSA (n = 5 each group, control = 0.88 % vs. 5azaD/TSA = 47.9%, p = 0.0009). In order to further confirm that in vivo administration of 5azaD/TSA is capable of augmenting the number of repopulating HSC, we performed serial transplantation studies. We observed that in vivo treatment of 5azaD/TSA of the primary host can cause successful human hematopoietic engraftment of the secondary (2 out of 4 mice) and tertiary hosts (2 out of 3 mice). These results are consistent with our earlier studies, in which ex-vivo treatment of CB HSC using 5azaD/TSA resulted in an absolute increase in the number of repopulating HSC which was associated with an increase in the transcript and protein levels of genes (HoxB4, Bmi-1 etc.) implicated in self-renewal of HSC (Araki et al. Blood 2007). In summary, our results indicate that in vivo intervention via epigenetic mechanisms may potentially provide new treatment modalities to boost the HSC pool in bone marrow failure or poor post transplant engraftment.