Chromatin-Modifying Agent-Expanded Human Cord Blood Cells Display Reduced Allostimulatory Capacity.

The limited number of hematopoietic stem cells (HSC) within a single unit of human cord blood currently limits its use as an alternate graft source. However, we have developed a strategy using 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), which expands transplantable HSC 7- to 10-fold. In our current studies, we have assessed the allostimulatory capacity of the 5azaD/TSA-expanded grafts. The coexpression of immunophenotypic dendritic cell (DC) markers, such as HLA-DR/CD86 and HLA-DR/CD11c as determined by flow cytometry, and the allostimulatory capacity of 5azaD/TSA-expanded cells as determined by MLC were both significantly lower than control. It has been previously demonstrated that STAT3 is indispensable for the differentiation of DC from HSC. Real-time quantitative PCR analysis revealed that 5azaD/TSA-expanded cells expressed more STAT3 transcript than control while also expressing increased transcripts for STAT3 inhibitors including SHP1, p21, and GATA1. Western blot analysis indicates that chromatin-modifying agent-expanded grafts displayed a reduced ratio of p-STAT3 to total STAT3 than control cultures, which is likely indicative of STAT3 inactivity in 5azD/TSA-expanded grafts. Culturing 5azaD/TSA-expanded cord blood cells in extended cultures reveals that they are still capable of generating DC. Notably, STAT3 inactivity was transient because the transcript levels of STAT3 and its inhibitors, including SHP1, were comparable between 5azaD/TSA and control cultures following extended culture. Taken together, our studies indicate that the reduced allostimulatory capacity of 5azaD/TSA-expanded cells is likely because of reversible inhibition of STAT3-dependent DC differentiation. These results suggest that a graft composed of 5azaD/TSA-expanded cells possesses relatively less allostimulatory response but is still capable of generating DC in permissive conditions.

Preclinical IV busulfan dose-finding study to induce reversible myeloablation in a non-human primate model.

In this study we utilized a large animal model to identify a dose of intravenous busulfan that can cause reversible myelosuppression. Nine baboons (Papio anubis) were treated with IV busulfan at 6.4 (Group A), 8 (Group B), or 9.6 mg/kg (Group C). Peripheral blood counts were measured up to 90 days after treatment and serial bone marrow samples were obtained to analyze CD34+ cell content and colony forming units. Overall, the highest grade of peripheral blood cytopenia was observed 15 days after treatment in all three groups (n = 3/group). In particular, we observed a notable reduction of neutrophil and platelet counts in the blood and the number of marrow CD34+ cells and colony forming units. In contrast, the effect of busulfan on hemoglobin levels was mild. Baboons who received the highest dose of busulfan showed only a 25-35% recovery of marrow CD34+ cells and colony forming units after 90 days of busulfan administration. However, all three groups of animals showed a full recovery of peripheral blood counts and normal marrow cellularity and tri-lineage hematopoiesis after treatment. Notably, all three doses of busulfan were tolerated well without significant extra-medullary toxicity. These results validate the hierarchy of blood cells likely targeted by busulfan, and based on these findings, clinical trials using myelotoxic but not myeloablative doses of intravenous busulfan will be designed for patients with myeloid malignancies.

Ex vivo expansion of human mobilized peripheral blood stem cells using epigenetic modifiers.

BACKGROUND: Epigenetic modifications likely control the fate of hematopoietic stem cells (HSCs). The chromatin-modifying agents (CMAs), 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), have previously been shown to expand HSCs from cord blood and marrow. Here we assessed whether CMA can also expand HSCs present in growth factor-mobilized human peripheral blood (MPB). STUDY DESIGN AND METHODS: 5azaD and TSA were sequentially added to CD34+ MPB cells in the presence of cytokines, and the cells were cultured for 9 days. RESULTS: After culture, a 3.6 ± 0.5-fold expansion of CD34+CD90+ cells, a 10.1 ± 0.5-fold expansion of primitive colony-forming unit (CFU)-mix, and a 2.2 ± 0.5-fold expansion of long-term cobblestone-area-forming cells (CAFCs) was observed in 5azaD/TSA-expanded cells. By contrast, cells cultured in cytokines without 5azaD/TSA displayed no expansion; rather, a reduction in CD34+CD90+ cells (0.7 ± 0.1-fold) and CAFCs (0.3 ± 0.1-fold) from their initial numbers was observed. Global hypomethylation corresponding with increased transcript levels of several genes implicated in HSC self-renewal, including HOXB4, GATA2, and EZH2, was observed in 5azaD/TSA-expanded MPB cells in contrast to controls. 5azaD/TSA-expanded MPB cells retained in vivo hematopoietic engraftment capacity. CONCLUSION: MPB CD34+ cells from donors can be expanded using 5azaD/TSA, and these expanded cells retain in vivo hematopoietic reconstitution capacity. This strategy may prove to be potentially useful to augment HSC numbers for patients who fail to mobilize.

Differential effects of epigenetic modifiers on the expansion and maintenance of human cord blood stem/progenitor cells.

Epigenetic therapies, including DNA methyltransferase and histone deacetylase (HDAC) inhibitors, are increasingly being considered to treat hematological malignancies, but their effects on normal hematopoietic stem cells (HSCs) remain largely unexplored. We compared the effects of several HDAC inhibitors, including valproic acid (VPA) and trichostatin A (TSA), alone or in combination with 5-aza-2'-deoxycytidine (5azaD) on the expansion of HSCs. VPA induced the highest expansion of CD34+CD90+ cells and progenitor cells compared with other HDAC inhibitors or the sequential addition of 5azaD/TSA in culture. Xenotransplantation studies demonstrated that VPA prevents HSC loss, whereas 5azaD/TSA treatment leads to a net expansion of HSCs that retain serial transplantation ability. 5azaD/TSA-mediated HSC expansion was associated with increased histone acetylation and transient DNA demethylation, which corresponded with higher gene transcript levels. However, some genes with increased transcript levels lacked changes in methylation. Importantly, a global microarray analysis revealed a set of differentially expressed genes in 5azaD/TSA- and VPA-expanded CD34+ cells that might be involved in the expansion and maintenance of transplantable HSCs, respectively. In summary, our data indicate that treatment of HSCs with different chromatin-modifying agents results in either the expansion or maintenance of HSCs, an observation of potential therapeutic importance.

Positive selection and transplantation of autologous highly purified CD133(+) stem cells in resistant/relapsed chronic lymphocytic leukemia patients results in rapid hematopoietic reconstitution without an adequate leukemic cell purging.

We assessed the capacity of positively selected autologous CD133(+) hematopoietic stem cells (HSCs) to reconstitute lymphomyelopoiesis in chronic lymphocytic leukemia (CLL) patients receiving myeloablative chemotherapy. Ten resistant/relapsed CLL patients underwent HSC mobilization with chemotherapy and granulocyte-colony stimulating factor (G-CSF). Positive selection of circulating CD133(+) HSCs was performed by immunomagnetic technique. Highly purified HSCs were reinfused after busulphan/melphalan myeloablative treatment. A median number of 4.2 x 10(6) CD34(+) cells/kg and of 3.14 x 10(6) CD133(+) cells/kg were collected. Immunomagnetic selection resulted in the reinfusion of a median number of 2.45 x 10(6) CD133(+) cells/kg (median purity: 94.8%; median recovery: 84%) and 2.4 x 10(6) CD34(+) cells/kg (median purity: 93%; median recovery: 71%). HSC selection resulted in a median T cell and CD19(+)/CD5(+) cell depletion of 3.85 log and 2.8 log, respectively. At the molecular level, however, 7 of 8 valuable purified HSC fractions were contaminated by leukemic cells. All CLL patients showed rapid and sustained myeloid engraftment after reinfusion of purified CD133(+) cells. Immunologic reconstitution was comparable to that routinely observed in patients reinfused with unmanipulated leukapheresis products and no late infectious complications were observed. With a median follow-up of 28 months for transplanted patients, 5 patients are in clinical complete remission, 3 are in partial remission, and 1 is in progression. In conclusion, the reinfusion of highly purified CD133(+) HSCs allowed the rapid and sustained recovery of hematopoiesis after myeloablative treatment in resistant/relapsed CLL patients. However, the purging potential of positive selection of CD133(+) cells is not adequate to achieve tumor-free autografts.