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.

Bone marrow CD34+ cells expanded on human brain endothelial cells reconstitute lethally irradiated baboons in a variable manner.

Increased cell dose has a positive impact on the therapeutic outcome of bone marrow (BM) hematopoietic stem cell (HSC) transplant. However, methods to successfully expand BM HSCs have yet to be achieved. It has been shown previously that ex vivo expansion of BM cells using porcine microvascular endothelial cells can rescue a baboon from a lethal dose of radiation. However, in a prior study, baboons that received CD34+ cell doses less than 4 x 10(6) cells/kg body weight failed to achieve hematopoietic reconstitution. In our present study we used human brain endothelial cells (HUBECs) and cytokines to expand BM cells, and examined their ability to provide hematopoietic reconstitution in three lethally irradiated baboons following autologous transplant as a surrogate preclinical model. After ex vivo culture, the grafts represented a 1.8- to 2.1-fold expansion of CD34+ cells, a 3.7- to 13.2-fold increase of colony-forming cells, and a 1.9- to 3.2-fold increase of cobblestone area-forming cells, in comparison to the input cell numbers. Despite transplanting CD34+ cell grafts displaying a comparable degree of expansion, there was an obvious variability in the kinetics of hematopoietic reconstitution. The variation in hematopoietic reconstitution cannot be fully explained by the properties tested in expanded CD34+ cells, and warrant caution against taking into account such attributes as cell dose, expression of adhesion molecules, and migration as a measure of successful expansion of HSCs.

Cord blood stem cell expansion is permissive to epigenetic regulation and environmental cues.

OBJECTIVE: Augmentation of the number of cord blood (CB) hematopoietic stem cells (HSC) present in a unit is required before it can be considered as an alternative graft for hematopoietic reconstitution for adult patients. In order to further optimize strategies to augment HSC numbers, we examined whether expansion of HSC mediated by epigenetic mechanisms remains permissive to external environmental cues. MATERIALS AND METHODS: The chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA) were used to ameliorate epigenetic alteration of CB cells during ex vivo culture by adding various cytokines. After culture, CD34(+)CD90(+) cell numbers, their division history, in vitro clonogenic potential, and in vivo hematopoietic reconstitution potential and frequency were determined. RESULTS: 5azaD/TSA-treated, CD34(+)CD90(+) cells were greatly influenced in terms of their degree of expansion, clonogenic potential, cell-division rate, and transplantability by the combination of cytokines used in culture. Furthermore, our current results verify that the sequential addition of 5azaD followed by TSA is crucial for expansion of HSC. We demonstrate that following 5azaD/TSA treatment, the rate of CD34(+)CD90(+) cell division is also dependent on the cytokine cocktail and that this is associated with functional changes, including alteration of in vitro clonogenic potential and in vivo reconstitution potential. CONCLUSIONS: Our studies indicate there are interactions between intrinsic factors influenced by epigenetic mechanisms and external environmental signals in the regulation of HSC expansion. Epigenetic influences on HSC can be accentuated by environmental factors. Regulation of the rate of divisions may be a critical determinant for the maintenance of HSC functional potency during ex vivo expansion.

Effect of periodontitis on insulin resistance and the onset of type 2 diabetes mellitus in Zucker diabetic fatty rats.

BACKGROUND: Studies indicate that an association exists between periodontitis and type 2 diabetes mellitus (T2DM) and/or obesity, with chronic inflammation hypothesized as the common denominator. The purpose of this study was to determine the causal effect of periodontitis and the concomitant impact of diet on the onset of insulin resistance (IR) and T2DM using a rat model system that simulates human obesity and T2DM. METHODS: Twenty-eight, 5-week-old female Zucker diabetic fatty (ZDF, fa/fa) rats were divided into four groups of seven animals: high-fat fed-periodontitis (HF/P), high-fat fed-no periodontitis (HF/C), low-fat fed-periodontitis (LF/P), and low-fat fed-no periodontitis (LF/C). Periodontitis was induced by ligature placement. Fasting plasma insulin and glucose levels were measured, and glucose tolerance tests were performed to assess glucose homeostasis, IR, and the onset of T2DM. The level of tumor necrosis factor-alpha (TNF-alpha), leptin, triglycerides, and free fatty acids were determined in week 13 at sacrifice. RESULTS: HF/P rats developed more severe IR compared to HF/C rats (P <0.01) and LF/P or LF/C rats (P <0.001) as measured by fasting insulin levels and homeostasis model assessment analysis. The onset of severe IR occurred approximately 3 weeks earlier in HF/P rats compared to HF/C rats. HF/P rats developed impaired (110 to 125 mg/dl) and frank fasting hyperglycemia (>125 mg/dl) 2 weeks earlier than HF/C rats. There was no difference in the severity and onset of IR and T2DM between LF/P and LF/C rats. The level of TNF-alpha was significantly higher in HF/P rats compared to HF/C rats (P <0.01). CONCLUSION: Periodontitis accelerated the onset of severe IR and impaired glucose homeostasis in high-fat fed ZDF rats.

Histochemical examination of periodontal junctional epithelium in p21/p27 double knockout mice.

The periodontal junctional epithelium (JE) is maintained in a steady state through a dynamic process that balances proliferation and exfoliation of epithelial cells. However, mechanisms that regulate JE are not well understood. To better understand how proliferation of the JE is controlled in healthy gingiva, we have studied functional roles of the CDK (cyclin dependent kinase) inhibitors p21 and p27 in JE using knockout mouse model systems. Image analysis of the dentogingival junction in p21 or p27 single knockout mice as well as p21/p27 double knockout mice (dKO) was performed. The analysis revealed enlarged JE in p21/p27 dKO mice due to an increase in the area of the epithelium and associated connective tissue 'islands'. Immunohistochemistry was performed for p21, p27, cyclin D1, and proliferating cell nuclear antigen (PCNA). The highest levels of PCNA-positive cells were detected in the p21/p27 dKO mice, reflecting increased cell turnover. Lower levels of cyclin D1 were detected in the JE of p21/p27 knockout mice, suggesting that p21 and p27 regulate stability of cyclin D1 in oral epithelium. These data suggest that p21 and p27 have a critical role in controlling epithelial cell proliferation in the JE and thus function to maintain the JE at a normal size.