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Strategies that increase homing to the bone marrow and engraftment efficacy of ex vivo expended CD34(+) cells are expected to enhance their clinical utility. Here we report that nicotinamide (NAM), a form of vitamin B-3, delayed differentiation and increased engraftment efficacy of cord blood-derived human CD34(+) cells cultured with cytokines. In the presence of NAM, the fraction of CD34(+)CD38(-) cells increased and the fraction of differentiated cells (CD14(+), CD11b(+), and CD11c(+)) decreased. CD34(+) cells cultured with NAM displayed increased migration toward stromal cell derived factor-1 and homed to the bone marrow with higher efficacy, thus contributing to their increased engraftment efficacy, which was maintained in competitive transplants with noncultured competitor cells. NAM is a known potent inhibitor of several classes of ribosylase enzymes that require NAD for their activity, as well as sirtuin (SIRT1), class III NAD(+)-dependent-histone-deacetylase. We demonstrated that EX-527, a specific inhibitor of SIRT1 catalytic activity, inhibited differentiation of CD34(+) cells similar to NAM, while specific inhibitors of NAD-ribosylase enzymes did not inhibit differentiation, suggesting that the NAM effect is SIRT1-specific. Our findings suggest a critical function of SIRT1 in the regulation of hematopoietic stem cell activity and imply the clinical utility of NAM for ex vivo expansion of functional CD34(+) cells., (Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Objective: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder associated with intravascular hemolysis and thrombosis. Hemolysis is caused by the hypersensitivity of PNH-red blood cells (RBC) to complement-mediated lysis due to deficiency in the surface glycosyl phosphatidylinositol-anchored antigens, CD55 and CD59. Thrombosis may be related to the platelet tendency to undergo hyperactivation. We previously suggested that hemolysis and thrombosis in other hemolytic anemias are related to oxidative stress. In the present study, we assessed the oxidative status of blood cells in PNH and tested the potential protective effects of antioxidants., Materials and Methods: Blood samples were obtained from 11 PNH patients and 11 normal control donors. Flow cytometry was used to measure oxidative stress markers in conjunction with the PNH immunophenotype., Results: Results indicated that abnormal, CD55/CD59-negative, RBC, neutrophils, and platelets are under oxidative stress. Their intracellular reactive oxygen species, membrane lipid peroxides, and external phosphatidylserine were higher and their reduced glutathione was lower than CD55/CD59-positive cells of the same patient or cells of normal controls. PNH-RBC were hypersensitive to an oxidative insult (e.g., hydrogen peroxide) and their oxidative status increased following interaction with complement, prior to hemolysis. Antioxidants reduced this hemolysis as well as activation of PNH platelets., Conclusion: We propose that oxidative stress mediates the symptoms of PNH and suggest that antioxidants might be considered as a therapeutic modality.

Hypoxia can induce erythropoiesis through regulated increase of erythropoietin (Epo) production. We investigated the direct influence of oxygen tension (pO(2)) in the physiologic range (2-8%) on erythroid progenitor cell differentiation using cultures of adult human hematopoietic progenitor cells exposed to decreasing (20% to 2%) pO(2) and independent of variation in Epo levels. Decreases in hemoglobin (Hb)-containing cells were observed at the end of the culture period with decreasing pO(2). This is due, in part, to a reduction in cell growth and, at 2% O(2), a marked increase in cell toxicity. Analysis of the kinetics of cell differentiation showed an increase in the proportion of cells with glycophorin-A expression and Hb accumulation at physiologic pO(2). Cells were characterized by an early induction of gamma-globin expression and a delay and reduction in peak levels of beta-globin expression. Overall, fetal Hb and gamma-globin expression were increased at physiologic pO(2), but these increases were reduced at 2% O(2) as cultures become cytotoxic. At reduced pO(2), induction of Epo-receptor (Epo-R) by Epo was decreased and delayed, analogous to the delay in beta-globin induction. The oxygen-dependent reduction of Epo-R can account for the associated cytotoxicity at 2% O(2). Epo induction of erythroid transcription factors, EKLF, GATA-1, and SCL/Tal-1, was also delayed and decreased at reduced pO(2), consistent with lower levels of Epo-R and resultant Epo signaling. These changes in Epo-R and globin gene expression raise the possibility that the early increase of gamma-globin is a consequence of reduced Epo signaling and a delay in induction of erythroid transcription factors.

Cell dedifferentiation occurs in different cell systems. In spite of a relative paucity of data it seems reasonable to assume that cell dedifferentiation exists in reversible equilibrium with differentiation, to which cells resort in response to intercellular signals. The current literature is indeed compatible with the concept that dedifferentiation is guided by structural rearrangements of nuclear chromatin, directed by epigenetic cell memory information available as silenced genes stored on heterochromatin, and that gene transcription exists in reversible "fluctuating continua" during parental cell cycles. Here, we review the molecular mechanisms of cell dedifferentiation and suggest for hematopoietic development that postnatal hemangioblasts are generated by dedifferentiation of committed hematopoietic stem cells.

Objectives: We have demonstrated epigenetic modulation of CD34(+) cell differentiation by the high-affinity copper (Cu) chelator tetraethylenepentamine (TEPA). TEPA slowed down the rate of CD34(+) cell differentiation and increased their engraftability in SCID mice. TEPA biological activity was attributed to its effect on cellular Cu levels as (a) treatment with TEPA resulted in reduction of cellular Cu, and (b) excess of Cu reversed TEPA's activity and accelerated differentiation. In the present study we further evaluated the role of cellular Cu in TEPA's biological activity., Methods: The effects of Cu-chloride, TEPA, TEPA/Cu mixtures at various ratios, and a synthesized, stable, TEPA-Cu complex on short- and long-term cord blood-derived CD34(+) cell cultures as well as on the overall and chelatable cellular Cu were investigated., Results: Addition of TEPA, TEPA/Cu mixtures at up to equimolar concentrations, and the TEPA-Cu complex to CD34(+) cell cultures resulted in inhibition of differentiation and enhancement of long-term self-renewal. Measurement of the overall cellular Cu by atomic absorption spectrophotometry showed 20 to 40% decrease by TEPA while the TEPA-Cu mixture and the TEPA-Cu complex increased cellular Cu by 10- to 20-fold, as did CuCl(2). However, measurement of the cellular pool of labile Cu showed similar reduction (50% from the control) by all the TEPA forms, while CuCl(2) increased it. Thus, inhibition of differentiation and enhancement of self-renewal of CD34(+) cells was correlated with reduction in the cellular chelatable Cu content., Conclusion: The results suggest that decreasing of the chelatable Cu pool, rather than overall Cu, is the mechanism that stands behind TEPA's biological activity.

Objective: For recipients of haploidentically mismatched stem cell allografts, T-cell depletion is mandatory to prevent lethal graft-vs-host disease (GVHD). Prevention of GVHD can be accomplished by negative selection of T cells or positive selection of stem cells. Recently, a new method for positive selection of stem cells was introduced using monoclonal antibodies against CD133 antigen. We report five cases of successful application of immunomagnetic separation of CD133+ stem cells for haploidentically mismatched allogeneic stem cell transplantation., Methods: Five patients with high-risk hematological malignancies, ages 7 to 63 years old (median, 17 years), underwent peripheral blood stem cell transplantation from haploidentically mismatched related donors. Conditioning protocol was tailored according to patient clinical situation and included combination of treosulfan/fludarabine/thiotepa/melphalan/Mabcampath. Two patients did not get thiotepa. One of them received a protocol that included infusion of 4.4 x 10(7) blood mononuclear cells from the donor (day -9), followed by a combination of fludarabine/cyclophosphamide/busulfex/MabCampath. Separation of CD133+ stem cells was done using CliniMACS with Miltenyi's CD133 reagent., Results: The procedure was well tolerated by all patients. Early 3-lineage engraftment was documented and none exhibited immune-mediated rejection. Time to recovery of absolute neutrophils count above 0.5 x 10(9)/L and 1.0 x 10(9)/L was 10 to 15 days (median, 14) and 11 to 29 days (median, 15), respectively. Time for platelet recovery to values greater than 20 x 10(9)/L and greater than 50 x 10(9)/L ranged from 12 to 25 days (median, 13.5), and from 14 to 34 days (median, 16), respectively. Transplant-related mortality did not occur in any of the patients., Conclusion: Our successful pilot trial suggests that positive selection of CD133+ stem cells may be a useful method for safe transplantation with haploidentically mismatched stem cell allografts while avoiding lethal acute and chronic GVHD. Future studies will be required to assess the clinical benefits of stem cell purification with CD133+ in comparison with CD34+ stem cells.

Objective: We previously demonstrated that cellular copper is involved in the regulation of proliferation and differentiation of hematopoietic progenitor cells. Modulation of cellular copper was achieved by supplementing the culture with a copper chelator that reduces cell copper content, or copper salts, which elevate the level of cellular copper. In the present study, we evaluated the effect of short-term (3-week) treatment with the copper chelator tetraethylenepentamine (TEPA) on short- and long-term (up to 11 weeks) ex vivo expansion of hematopoietic progenitors, as well as on their SCID engraftment potential., Materials and Methods: Cord blood-derived purified CD34+ cells were grown in liquid medium supplemented with the cytokines stem cell factor, thrombopoietin, Flt3 ligand, and IL-6, and the chelator TEPA for the first 3 weeks and then for up to 11 weeks with cytokines alone. Control cultures were supplemented with cytokines alone for the entire culture duration. Cultured cells were characterized by immunophenotyping and cloning (CFUc). Transplantability was assayed by injection of repurified CD34+ cells into NOD/SCID mice., Results: In the short term, TEPA supported increased percentages of early progenitors over control cultures incubated with cytokines alone (CD34(+)CD38-, p=0.001 and CD34(+)Lin-, p=0.016). In the long term, TEPA pretreated cultures showed prolonged expansion of CD34+ cells (p=0.01) and CFUc (p=0.002) compared with that of untreated cultures. The SCID engraftment potential of CD34+ cells repurified from the TEPA-treated cultures was higher compared with that of the control, i.e., only cytokine-treated cultures (p=0.03)., Conclusion: TEPA enabled preferential proliferation of early progenitor cells with the phenotype CD34(+)CD38- and CD34(+)CD38- Lin- during the first weeks of culture, resulting in the observed increased long-term ex vivo expansion and engraftment capabilities.

The pathophysiology of sickle cell anemia is primarily explained in terms of the oxygen-dependent polymerization of sickle hemoglobin (HbS) followed by sickling of erythrocytes. Since the rate and extent of HbS polymerization depend on its intracellular concentration, it has been generally assumed that sickling occurs primarily in mature erythrocytes with their high intracellular hemoglobin concentration. In the present study, we investigated the propensity of nucleated erythroid precursors to undergo sickling; both cultured and fresh marrow-derived erythroid precursors from patients with homozygous sickle cell anemia were studied. The results revealed that upon deoxygenation cultured erythroblasts underwent characteristic morphological deformation in the form of fine, fragile, elongated spicules. Ultrastructural analysis demonstrated highly organized and tightly aligned hemoglobin fibers in the protruded regions. Bone marrow cells examined under partial or complete deoxygenated conditions displayed similar morphological changes. When cultured SS erythroid precursors were exposed to hydroxyurea or butyrate, drugs that may increase fetal hemoglobin (HbF) and inhibit intracellular polymerization, a significant decrease was observed in the propensity of these precursors to undergo sickling, accompanied by a three- to fivefold increase in HbF. These results suggest that, in addition to mature erythrocytes, nucleated erythroid precursors in the bone marrow have the capacity to undergo characteristic sickling as a result of HbS polymerization and may be involved in several aspects of the pathophysiology of sickle cell anemia. Treatment with HbF-stimulating drugs may benefit patients with this disease by inhibiting polymerization-induced sickling of erythroid precursors in the marrow as well as mature erythrocytes in the peripheral blood.

The cell cycle status of human erythroid precursors generated in a two-step liquid culture was studied by a double-labeling flow cytometric technique. Following a first phase, where peripheral blood mononuclear cells were cultured in the presence of a combination of growth factors, not including erythropoietin (Epo), the cells were washed and recultured in a second phase in the presence of Epo. This procedure resulted in a stimulation of the proliferation and maturation of erythroid precursors. In the presence of optimal concentrations of Epo (2 U/mL), a high percentage (> 40%) of cells were found in the S phase of the cell cycle until day 10. Then, as a result of maturation, the proportion of cells in S gradually decreased, reaching less than 2.0% by day 21. At this time, the culture consisted of > 95% hemoglobin-containing, nonproliferating, orthochromatic normoblasts. Cell cycle analysis of this normoblast population demonstrated a bimodal distribution; while the majority of the cells had a diploid (2C) DNA content, i.e., cells in G1 (or G0) phase, a sizable fraction was tetraploid (4C) corresponding to cells in G2. In contrast, in cultures stimulated with physiological concentrations of Epo (around 50 mU/mL), all the terminally differentiated cells were arrested at the G1 phase. These results suggest that Epo is an essential growth-promoting factor for erythroid precursors, but supraphysiological concentrations, such as present in vivo in severe anemia (e.g., aplastic anemia) or after Epo administration, may be associated with development of normoblasts with abnormal DNA content.

We have recently described a two-step liquid culture system that supports the proliferation and maturation of human erythroid progenitors. Several days after the addition of erythropoietin, the cultures undergo erythroid differentiation in a synchronized fashion. The purpose of the present study was to determine detailed kinetics of globin gene expression at the mRNA level in adult and newborn erythroid cells. Our results show that in cultures derived from normal adult peripheral blood, the mRNA levels of alpha- and beta-globin genes increased throughout most of the culture period, whereas gamma-globin mRNA remained at a low level. In contrast, high expression of all three globin genes, alpha, beta, and gamma, was observed in cultures derived from cord blood. The results demonstrate that the populations of erythroid progenitors in cord blood and in adult peripheral blood are fundamentally different, suggesting that this culture system recapitulates the normal pattern of globin gene expression, providing a valuable tool in the investigation of the regulation of the switch from fetal to adult hemoglobin.

Irradiation with long-wave UV light (LUV) at 366 nm of cells that had been incubated with 12-(1-pyrene)dodecanoic acid (P12), a fatty acid derivative with a covalently linked pyrene nucleus, resulted in cytotoxicity. Using the in vitro established human cell lines HL-60 and U-937, we demonstrated that these leukemic cells are much more susceptible to the photosensitizing effect of P12 than normal bone marrow (BM); a 4-log reduction in the number of clonogenic leukemic cells was achieved under conditions where colony formation by normal hemopoietic progenitors was reduced by less than 40%. Moreover, the results of irradiating mixed populations of leukemic and normal cells indicated that phototoxicity of leukemic cells was not affected by the presence of a large excess of normal BM cells, nor was the survival of normal BM cells influenced by the presence of leukemic cells. These findings suggested that the procedure could be adapted for selective ex vivo elimination of malignant cells, i.e., purging of BM in remission prior to autologous transplantation.

A new myelomonoblastic cell line (M20) was established from the peripheral blood of a ten-year-old child with acute myeloblastic leukemia, using an improved method for supporting the initial stages of cell proliferation. The addition of irradiated macrophage monolayers to the proliferating cells appeared to overcome the deterioration of the primary cultures and enable them to continue proliferating until they became independent of this environment. The cell line that developed consisted of myeloblasts and promyelocytes characterized by light and scanning electron microscopy, cytochemistry, and enzymatic activities. The cells expressed Fc receptors and WT1 antigens but did not exhibit HLA-DR, HMA1, Epstein-Barr virus nuclear antigen, and surface Ig. The M20 cells produced colonies when cultured in semisolid medium and secreted lysozyme, prostaglandin E2, and interleukin 1. An attempt was also made to analyse the position of the M20 cells in the scheme of differentiation of the myelomonocytic lineage using different approaches. Treatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate induced their adherence to plastic surfaces and partial maturation to macrophages as judged by morphological criteria, cytochemistry, and enzyme activities. However, comparison of the M20 cells to other well-established myelomonoblastic cell lines did not reveal any pattern suggesting a possible relationship between surface markers, cell function, and differentiation pathway of the various cell lines tested. Establishment of additional cell lines and identification of new markers may assist in defining the mechanisms involved in normal differentiation and malignant transformation of this cell lineage. In addition, such cell lines may also provide a tool for the quantitative recovery of a variety of monokines.

The presence of myelomonocytic progenitor cells in human peripheral blood was used for the analysis of cloned populations of human monocytes. Colonies of granulocytes and macrophages were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by nonstimulated PBM (CM). Following 20-25 days of incubation, most colonies were found to consist of cells with monocyte-macrophage morphology. Cloned populations of monocytes were tested for several monocyte membrane markers and compared to noncloned adherent monocytes. HLA-DR, 63D3, LeuM2 antigens and Fc receptors were expressed on cells from individual colonies in similar proportions to their expression on noncloned monocytes. Some colonies were uniform in their negative expression of the 63D3 antigen, as were the noncloned monocytes. Although the clonality of cells tested was not directly proven, these results indicated that at least for some monocyte markers, heterogeneous expression was obtained in monoclonal populations of monocytes. It is possible, however, that testing of additional markers and functions may reveal homogeneous clones of monocytes and suggest the existence of stable subsets.