ALBERT

Description: Recently, we have demonstrated that modifications of chromatin involving DNA methylation and histone acetylation events can change the fate of hematopoietic stem cells (HSC), likely as a result of altered gene expression patterns (Araki H et al. Blood 2007). In our current studies we have examined the epigenetic regulation of genes induced during the expansion of HSC by chromatin modifying agents [5aza-2-deoxycytidine (5azaD), trichostatin A (TSA)]. The expression of the polycomb repressing complex gene, enhancer of zeste homolog 2 (Ezh2) has been implicated in histone methylation and deacetylation. We observed a significant increase in the transcript levels of Ezh2 in addition to self-renewal genes HoxB4, Bmi1 and GATA-2 in 5azaD/TSA treated expanded human cord blood (CB) cells. Ezh2 overexpression has been shown to prevent HSC exhaustion in a murine model by chromatin stabilization. CB CD34+ cells expanded in 5azaD/TSA resulted in retention of relatively higher transcript levels of DNA methyltransferase (DNMT), DNMT1 and lower levels of DNMT3a and DNMT3b in CD34+CD90+ cells, the most primitive subpopulation, in contrast to the relatively mature CD34−CD90− cells. The retention of higher DNMT1, the maintenance methyltransferase, in the most primitive CD34+CD90+ cells may favor the chromosomal stability of expanded grafts. Cytogenetic analysis and DNA ploidy of expanded grafts were normal and the telomere length of expanded CB grafts did not reveal any significant alteration of their lengths in comparison to unmanipulated primary CB grafts. In order to explore the roles of epigenetic modifications on the differentiation of HSC during ex vivo expansion, we have also examined the transcript levels of genes associated with differentiation in 5azaD/TSA expanded cells in comparison to cells expanded in cytokines alone. Interestingly, the transcript levels of differentiation associated genes Pu.1 and GATA-1 decreased following 5azaD/TSA treatment. The lower transcript levels of differentiation inducing genes (Pu.1, GATA-1) and higher levels of self-renewing genes (Ezh2, HoxB4) in 5azaD/TSA expanded cells suggest a possible interaction among these functionally opposing groups of genes which are likely regulated by epigenetic mechanisms. By using a chromatin immunoprecipitation (ChIP) assay we observed significantly increased levels of histone H4 acetylation on the promoter sites of Hox-B4, Bmi-1, and GATA-2 and decreased levels of acetylation on the promoter sites of genes involved in differentiation (Pu.1, GATA-1) in 5azaD/TSA treated CB cells. The degree of histone H4 acetylation of these promoters sites directly correlated with the transcript levels of these genes as evidenced by real time quantitative PCR. Futhermore, silencing of HoxB4 by using short hairpin RNA (shRNA) during 5azaD/TSA mediated expansion resulted in about a 50% reduction in the expansion of CB cells in contrast to cells containing non-silencing control shRNA, indicating HoxB4 is required for HSC expansion. Understanding how the possible interactions between functionally opposing groups of genes regulating the expansion and differentiation of HSC is mediated by epigenetic mechanisms may further facilitate the optimization of ex vivo expansion strategies of CB grafts for therapeutic use.

Description: Abstract 2994 The suboptimal numbers of hematopoietic stem cells (HSC) present in human umbilical cord blood (CB) grafts results in a significant delay in blood cell reconstitution or graft failure, particularly in adult patients following transplantation. Using chromatin modifying agents (CMAs), including 5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA), we developed an ex-vivo expansion strategy that permits 7-fold expansion of transplantable HSC (Araki et al. Blood 2007, Exp Hematol 2009). Here we performed validation studies in non-human primate (NHP) baboons to examine the potential genotoxicity of CMA-expanded bone marrow (BM) graft. To our knowledge, there have been no reports suggesting 5azaD exacerbate chromosomal instability in human. We have demonstrated that the sequential addition of low-dose 5azaD (5 micromolar) and TSA (5 ng/ml) to human CB CD34+ cells does not result in a persistent depletion of Dnmt1 transcripts and protein levels. In this global methylation analysis using a LINE-1 assay indicates that DNA demethylation, which occurs with CMA treatment in culture, is transient: LINE-1 methylation of CD34+ cells in the absence or presence of CMA was 78.5% ± 2.2% and 55.4 % ± 2.2% respectively at day 3 while methylation levels of treated CD34+ cells returns to 69.7% ± 2.1% by day 9. In an attempt to evaluate the genotoxic potential of low-dose 5azaD/TSA, we transplanted baboons with CMA-expanded autologous BM grafts following a myeloablative dose of IV busulfan (4 mg/kg/day × 4 days). Although the initial pre-expansion CD34+ cell dose of CMA-expanded BM grafts was low (0.68, 2.64 and 5.18 × 106 CD34+ cells/kg respectively), all three baboons engrafted following transplantation. Following peripheral blood (PB) cell count recovery we examined BM CD34 and colony-forming cell (CFC) counts. Of note, the absolute number of CD34+ cells in the BM following hematopoietic reconstitution (926.0 ± 142.8 /microliter) was 3.5 fold higher than pre-transplant levels (257.9 ± 56.2 /microliter). However, the absolute number of CFC in the BM was 2.2 fold lower than pre-transplant levels. BM biopsies performed on the 3 baboons serially prior to, and 56, 120 and 400 days post-transplant respectively to examine hematopoietic recovery, myeloid-to-erythroid ratio and cellularity did not demonstrate myelodysplasia or malignancy. There was tri-lineage hematopoiesis evident in the BM in all 3 baboons with 50 to 70% cellularity. Further evaluation of the safety of 5azaD/TSA-expanded grafts was performed using three independent methods. Firstly, 150 NOD/SCID mice who received 5azaD/TSA expanded human CB grafts have not displayed any signs of tumor formation after primary or secondary transplantation. Secondly, karyotyping of CMA-expanded human CB graft or baboon CD34+ cells prior to and following transplant (day 266) reveals no detectable chromosomal anomalies. Thirdly, we used Flow-FISH, an in situ flow cytometry-based assay to measure relative telomere lengths (RTL) of purified CD11a+ (97.59% ± 0.38%) leukocytes from baboon BM prior to and following transplantation. The majority of CD11a+ leukocytes are short-lived which indirectly reflects the telomere lengths of the respective HSC from which they are derived. Our results show that the RTL of Jurkat cells were 10.6% ± 0.37%, while human CB cell RTLs were 19.7% ± 1.2%. The pre- and post-transplant (day 56) BM cells of one baboon (PA7619) displayed (average) RTL values of 13.48% ± 0.76 % and 13.96% ± 0.68% respectively. In another animal (PA7963), average RTL values were determined serially using Flow-FISH on BM CD11a+ enriched cells at multiple time-points post-transplant up to day 417 which displayed mean RTL of 17.66 % ± 0.52% while pre-transplant RTL was 15.39% ± 0.62%. In summary, the mean RTL values of baboon leukocytes were stable and post-transplant values were comparable to pre-transplant levels. Importantly, year-long monitoring shows no evidence of hematologic malignancies as determined by PB counts, BM examination (including cytogenetics) and measurements of leukocyte RTL values in a clinically relevant NHP model. These studies offer preliminary evidence that CMA-mediated expansion of CD34+ cells transferred into primates (and human CB CD34+ cells transferred into mice) is not genotoxic and does not induce hematopoietic malignancies, of relevance to use of human CMA-expanded CB grafts in clinical trials. Disclosures: No relevant conflicts of interest to declare.

Description: The lack of optimal number of repopulating hematopoietic stem cells (HSC) and graft failure following transplantation remain as obstacles to using cord blood (CB) as a true alternative graft for adult patients. Using chromatin modifying agents {5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA)}, we have developed a culture regimen that permits expansion of primitive CB CD34+ CD90+ cells resulting in a 10 fold expansion of in vivo repopulating cells without jeopardizing their multilineage differentiation capacity. Our results also indicate that the chromatin modifying agents likely mediate this function by inducing expression of endogenous genes (HoxB4, Bmi-1, GATA-2, etc.) previously implicated in self-renewal of HSC (Araki H et al. Blood2007:109:3670–3678). In our current studies we have further characterized the allostimulatory function of these expanded CB to assess its potential as grafts to mount an immune response. The percentage of 5azaD/TSA treated expanded CB cells co-expressing HLA-DR and CD86, or CD11c was less than cells expanded in cytokines alone. The expression of HLA-DR/CD40 was not altered following 5azaD/TSA treatment. In a mixed lymphocyte reaction (MLR), the irradiated CB cells (stimulator) were co-cultured with allogeneic peripheral blood T cells (responder) at 1:2 ratios. The MLR results indicate that 5azaD/TSA treated expanded CB grafts possessed diminished allostimulatory response as revealed by their relatively lower stimulation index in comparison to cells expanded in cytokines alone. Interestingly, the stimulation index of 5azaD/TSA treated cells (day 9) was even less than unmanipulated primary CB CD34+ cells (day 0). The reduced allostimulatory capacity of 5azaD/TSA expanded CB cells may favor in the reduction of incidence of graft rejection / graft failure following infusion. Alterations in cell fate regulatory mechanisms raise concerns for tumorigenesis, so the safety of such grafts was further assessed by examining the telomere length and cytogenetic analysis. Both cytogenetic analysis and examination of cell-cycle showed normal karyotype and diploid nature of expanded grafts, respectively. The telomere length of ex vivo expanded grafts as a putative indicator for their proliferative potential did not reveal significant alteration in their length in comparison to unmanipulated CB grafts. Also, in vivo repopulation assays with xenogeneic NOD/SCID mouse model showed no apparent tumor development following transplantation of ex vivo expanded 5azaD/TSA treated CB cells. In summary, our results indicate that the chromatin modifying agents not only result in expansion of in vivo repopulating CB cells but are capable of suppressing immunostimulatory capacity of expanded grafts and these grafts retain their telomere length and normal ploidy and can be expanded from a single unit for potential clinical use.

Description: Idiopathic myelofibrosis (IM) is characterized by the constitutive mobilization of CD34+ cells. IM peripheral blood (PB) CD34+ cells had a reduced cloning efficiency and a lower frequency of cobblestone areas compared with normal granulocyte colony-stimulating factor (G-CSF)-mobilized PB CD34+ cells. IM CD34+ cells engrafted nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, demonstrating that they contain bone marrow (BM)-repopulating cells. G-CSF-mobilized CD34+ cells produced multiple hematopoietic lineages within the NOD/SCID mice with a predominance of CD19+ cells. By contrast, IM CD34+ cells produced predominantly CD33+ cells, increased numbers of CD41+ cells, but fewer CD19+ cells. Transcriptional clonality assays of the engrafted human IM cells demonstrated their clonal origin. CD34+ cells from one patient isolated prior to leukemic transformation were capable of generating acute leukemia in NOD/SCID mice. The engrafted human cells exhibited the same abnormal karyotype as primary cells in a portion of the population. These findings demonstrate that BM-repopulating cells and more differentiated progenitor cells are constitutively mobilized into the PB in IM, and that their differentiation program is abnormal. In addition, the NOD/SCID model may be useful in gaining an understanding of the events occurring during the transition of IM to acute leukemia. (Blood. 2005;105:1699-1705)