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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.)

Iron is important for many biological processes, and its deficiency or excess is involved in pathological conditions. Although most iron is firmly bound (e.g., in hemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The level of LIP is regulated to meet the cell's requirements for iron but prevent excess. We describe herein a multiparameter flow cytometry procedure for measuring LIP in various human hematopoietic cells. Peripheral blood and bone marrow (BM) cells were loaded with calcein-AM, washed, and then incubated with or without the high-affinity iron-chelator Deferiprone (L1). Specific cell subpopulations were identified based on side-light scattering and expression of surface antigens. LIP was determined based on the ability of L1 to bind and remove iron from calcein and thereby increase the fluorescence emitted by the cells. Blood cells differ in their LIP content in the order monocytes > PMN > RBC > lymphocytes. Analysis of BM cells indicated a similar tendency among precursors of the different lineages. The results also showed that among myeloid precursors, LIP increases along cell maturation. Flow cytometry might be useful for evaluating LIP in various diseases and for studying the efficacy of iron-chelators., ((c) 2007 International Society for Analytical Cytology)

Clinical observations suggest that copper (Cu) plays a role in regulating hematopoietic progenitor cell (HPC) development. Cu is known to generate oxidative stress in cells which in turn affects proliferation, differentiation and apoptosis. To study this role of Cu, we used double staining flow cytometry to measure reactive oxygen species (ROS) generation by neonatal cord blood-derived CD34(+)CD38(-) cells. ROS was increased by Cu and was decreased by the Cu chelator tetraethylenepentamine (TEPA). Previously, we showed that TEPA reduces the free Cu content of HPCs and stimulates their ex vivo expansion. The present results suggest that TEPA affects expansion of HPCs by lowering their oxidative stress.

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: 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 CD34+ CD38- subset of human hematopoietic stem cells are crucial for long-term ex-vivo expansion; conditions that decreased this specific sub-population reduced the self-renewal capacity and shortened the duration of the proliferative phase of the culture. Retinoids, such as all-trans retinoic acid (ATRA), have been shown to induce CD38 expression. ATRA present in serum may be responsible for the high CD38 of cells grown in serum-containing medium. In the present study we analyzed the effects of AGN 194310, a retinoic acid receptor pan-antagonist, on CD38 expression of human hematopoietic cells. Normal cells (cord blood derived CD34+ cells) and abnormal cells (myeloid leukemic lines) were studied when grown in either serum-containing or serum-free media. The results showed that both serum and ATRA enhanced differentiation and, thereby, reduced the proportion of CD34+ CD38- cells and total CD34+ cell expansion. AGN reversed these effects of serum and ATRA: it delayed differentiation and increased CD34+ CD38- cells. These results suggest that physiological ATRA levels in serum may prevent efficient cell expansion. AGN, by neutralizing ATRA, improves cell expansion in serum-containing cultures, thus making AGN a useful agent for ex vivo expansion of stem cells and other specific sub-populations for research and clinical use.

We have previously found that the synthetic polyamine tetraethylenepentamine (TEPA) significantly delayed differentiation and prolonged expansion of cord-blood derived HPC in cytokine-supplemented cultures. Most HPC have the CD34+CD38+ phenotype, but the minority CD34+38- cells are primitive subset of HPC that have the potential for long-term repopulation in vivo. We investigated the effect of TEPA on the CD34/CD38 surface antigen expression of human myeloid leukemia cell lines as well as normal cord blood derived hematopoietic cells. Confirming previous results, our data showed that both the leukemic and normal cells increased their CD38 expression when grown in serum-containing medium or when treated with retinoic acid. In the present study, we found that TEPA inhibited CD38 under these conditions in both normal and leukemic cells. As for CD34, TEPA increased the proportion of CD34 cells in short- and long-term normal cultures but not in the leukemic cell lines. These results suggest that ex vivo expansion of HPC depends on the presence of CD34+CD38- cells and that TEPA prolongs HPC expansion by inhibiting the CD38- to CD38+ transition.

Background: We have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+CD38-, CD34+CD38-Lin-) in human umbilical cord blood (CB)-derived CD34+ cell cultures. This study extends our previous findings that copper chelation can modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells., Methods: In the present study we established a clinically applicative protocol for large-scale ex vivo expansion of CB-derived progenitors. Briefly, CD133+ cells, purified from CB using Miltenyi Biotec's (Bergisch Gladbach, Germany) CliniMACS separation device and the anti-CD133 reagent, were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). This protocol was evaluated using frozen units derived from accredited cord blood banks., Results: Following 3 weeks of expansion under large-scale culture conditions that were suitable for clinical manufacturing, the median output value of CD34+ cells increase by 89-fold, CD34+CD38- increase by 30-fold and CFU cells (CFUc) by 172-fold over the input value. Transplantation into sublethally irradiated non-obese diabetic (NOD/SCID) mice indicated that the engraftment potential of the ex vivo expanded CD133+ cells was significantly superior to that of unexpanded cells: 60+/-5.5% vs. 21+/-3.5% CD45+ cells, P=0.001, and 11+/-1.8% vs. 4+/-0.68% CD45+CD34+ cells, P=0.012, n=32, respectively., Discussion: Based on these large-scale experiments, the chelator-based ex vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing CB transplantation for hematological malignancies.

We examined the influence of cytokines on erythroid- and myeloid-lineage development of AC133+ cells during primary and secondary cultures. Cells cultured for 14 d in liquid medium containing erythropoietin (EPO) were amplified 831-fold with 98.2% erythroid cells. A similar culture exposed to granulocyte colony-stimulating factor (G-CSF) grew 1350-fold with 97.4% myeloid cells. To assess whether the cells with EPO inducement could respond at this point to G-CSF signal, or vice versa, the EPO-stimulated population was re-grown with G-CSF, constituting 95.2% myeloid, of 5075-fold, cells after 14 d of re-culture. Conversely, reculture of the G-CSF-stimulated population with EPO resulted in a 4083-fold growth with 81.4% erythroid cells. Semisolid culture containing EPO orG-CSF showed that some individual colonies had self- renewal potential after 14 d culture and could be induced todevelop into a different lineage. Analysis of primitive markers, CD34 and Notch1, or lineage markers, EPO-R and CD13, by single-cell reverse transcription polymerase chain reaction showed that individual colonies of 2-16 cells contained at least one CD34-positive cell with expression ofNotch1 and co-expression of EPO-R and CD13 appeared on either CD34-positive or CD34-negative cells. In situ hybridization with the same cell surface markers in cell populations confirmed the asymmetric cell division and co-expression from single cell data. The study provides a useful model for the analysis of multipotential progenitor development, and indicates that progenitor cells co-express genes from different lineage pathways before commitment and that cytokines influence lineage commitment.

Several clinical observations have suggested that copper (Cu) plays a role in regulating haematopoietic progenitor cell (HPC) development. To further study this role we used an ex vivo system. Cord blood-derived CD34+ cells were cultured in liquid medium supplemented with Kit- ligand, FLt3, interleukin 6 (IL-6), thrombopoietin and IL-3. Under these conditions, Cu content, measured by atomic absorption, was 7 ng/10(7) cells. Modulation of intracellular Cu was achieved by supplementing the cultures with the Cu chelator tetraethylenepentamine, which reduced cellular Cu (4 ng/10(7) cells), or ceruloplasmin or Cu sulphate that elevated cellular Cu (18 and 14 ng/10(7) cells respectively). The results indicated that low Cu content delayed differentiation, as measured by the surface antigens CD34, CD14 and CD15, colony-forming unit (CFU) frequency and cell morphology, while high Cu accelerated differentiation compared with Cu unmanipulated cultures. As a result, expansion of total cells, CFU and CD34+ cells in low Cu was extended (12-16 weeks), and in high Cu was shortened (2-4 weeks), compared with control cultures (6-8 weeks). These effects required modulation of intracellular Cu only during the first 1-3 weeks of the culture; the long-term effects persisted thereafter, suggesting that the decision process for either self-renewal or differentiation is taken early during the culture. This novel method of controlling cell proliferation and differentiation by copper and copper chelators might be utilized for ex vivo manipulation of HPC for various clinical applications.

In both sickle cell (SS) anemia and beta-thalassemia (beta-thal), an increase in fetal hemoglobin (HbF) ameliorates the clinical symptoms of the underlying disease. Several pharmacologic agents have been used to elevate HbF levels in adults; however, concerns regarding adverse effects of the prevailing drugs raise an urgent need for other agents capable of stimulating HbF production. We show here that sodium phenylacetate (NaPA) and its precursor, sodium 4-phenylbutyrate (NaPB), can enhance HbF production in cultured erythroid progenitor derived from normal donors and patients with SS anemia or beta-thal, when used at pharmacologic concentrations. Treatment resulted in (1) reduced cell proliferation, (2) elevated hemoglobin (Hb) content per cell (mean cellular Hb [MCH]), and (3) an increased proportion of HbF produced, associated with elevated levels of gamma-globin mRNA. Moreover, the active phenyl-fatty acids, with NaPA as a prototype, potentiated HbF induction by other drugs of clinical interest, including hydroxyurea (HU), sodium butyrate, and 5-azacytidine (5AzaC). Efficacy could be further enhanced by introducing chlorine substituents at the phenyl ring to increase drug lipophilicity. Our findings indicate that NaPA and NaPB, both already proven safe and effective in treatment of children with urea cycle disorders, might benefit also patients with severe hemoglobinopathies. The two-phase liquid culture procedure used in this study should prove valuable in further studies exploring the mechanisms of HbF induction by these agents, and might provide an assay to predict patient response in the clinical setting.

This study aimed to assess the effect of the M20 interleukin-1 (IL-1) inhibitor on normal and leukemic hematopoietic cells. The M20-derived IL-1 inhibitor was found to inhibit the growth of various hematopoietic cells. The in vitro proliferation of myeloid cell lines in serum-containing medium or proliferation of these cells induced by IL-1 in serum-free medium (measured by 3H-TdR) were inhibited by the M20 IL-1 inhibitor. In addition, growth of normal progenitors and fresh leukemic cells stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) (as measured by colony and liquid systems) was also inhibited by this factor. After the removal of the IL-1 inhibitor at the peak of growth inhibition, leukemic and normal progenitor cells retain their ability to grow and develop into GM-CSF colonies. These results show that the growth inhibition phenomena were reversible and did not result from a cytotoxic effect. Our data suggest that the M20-derived IL-1 inhibitor might function as a true negative growth regulator of normal and leukemic hematopoietic cells.

We have recently described a new two-phase liquid culture that supports the development of human erythroid progenitors (Fibach et al., Blood 73:100, 1989). The procedure separates the erythroid burst-forming units (BFUe) from the erythroid colony-forming units (CFUe) stage and enables quantitation of the proliferation and differentiation of BFUe into CFUe. In the present study we have utilized this system to study erythroid progenitors in polycythemia vera (PV). The abnormality of the erythroid series in PV has been shown to be associated with an increased responsiveness of the progenitors to the hormone erythropoietin (Epo). A basic question in this clonal stem cell disorder is at what developmental stage this abnormality of the PV clone is phenotypically expressed. We have studied this question by comparing the development of Epo-dependent and Epo-independent CFUe from peripheral blood BFUe of the PV patient during the BFUe to CFUe transition in the liquid culture. The results indicated that both types of CFUe are generated and that in all cases tested the ratio of Epo-independent progenitors at both the BFUe and CFUe stage was similar indicating no preferential development of Epo-independent CFUe. These results suggest that the abnormality of the PV erythroid progenitors is expressed only at the CFUe level. Moreover, since the liquid culture did not contain Epo, the results also support the conclusion that BFUe do not require Epo for proliferation or differentiation into CFUe.

Osteopetrosis is a congenital disease in which the bone marrow is replaced by osteoid, resulting in secondary extramedullary hematopoiesis. In 3 patients with osteopetrosis, circulating myeloid and erythroid progenitors were assayed. Erythroid burst-forming units were increased above normal by a factor of five and the myeloid colony-forming units were increased by a factor of ten. Myeloid cluster-forming units and erythroid colony-forming units, the more differentiated progenitor cells normally found only in the bone marrow, could also be detected in the peripheral blood of the 3 patients. The appearance of the differentiated progenitors in the peripheral blood suggests that extramedullary hematopoietic sites, in contrast to the bone marrow, may release progenitor cells more readily into the circulation. If this is the case, culturing progenitor cells may provide a sensitive noninvasive laboratory test to determine the presence of extramedullary hematopoiesis.

The ability of circulating progenitor cells from patients with polycythaemia vera (PV) and myelofibrosis with myeloid metaplasia (MMM) to develop erythroid colonies was studied in cultures with and without erythropoietin. In all normal controls, patients with secondary polycythaemia and MMM, erythroid colonies developed only after the addition of erythropoietin. Only in patients with PV, both in the active and spent phases of the disease, erythroid colonies developed in the absence of erythropoietin. The results indicate the perpetuation of erythropoietin-dependent, as well as erythropoietin-independent progenitors in both phases of this disease. Although spent PV often clinically resembles MMM, there is a basic difference in the behaviour of the circulating erythroid progenitors in these diseases which may serve as a useful tool in discriminating MMM from spent PV, when there is no history of active PV.

Hemopoiesis is studied in vitro mainly in semisolid culture, where hemopoietic progenitors develop into discrete colonies. We describe a liquid culture system that supports the proliferation and maturation of human erythroid progenitors. We seeded mononuclear cells from the peripheral blood (PB) of patients with beta-thalassemia in liquid medium in the presence of conditioned medium from human bladder carcinoma cells. Seven days later, RBCs, normoblasts, granulocytes, and monocytes disappeared, and the number of lymphocytes dropped considerably. In contrast, erythroid colony-forming cells increased fourfold to tenfold. The next step entailed the removal of colony-stimulating factor (CSF) and CSF-secreting cells, the exclusion of macrophages by harvesting nonadherent cells, and the lysis of T lymphocytes by treatment with monoclonal rat antihuman lymphocyte antibodies (CAMPATH-1) and complement. Reculture of the remaining cells in liquid medium supplemented with recombinant erythropoietin (EPO) resulted in the exclusive development of erythroid cells, with myeloid cells reduced to less than 2%. Stainable hemoglobin (Hb) appeared on day 3, with over 85% of the population containing hemoglobin by day 11 and the cell number increasing from 0.2 X 10(6) to 3 X 10(6) mL. By permitting the manipulation of culture conditions and components and increasing the cell yield, the liquid system may facilitate quantitative analysis of growth kinetics as well as biochemical and immunologic characterization of the developing erythroid cell.

Analysis of methylation at the beta-globin gene cluster was carried out on DNA derived from nucleated RBCs (orthochromatic normoblasts) isolated from peripheral blood of patients with beta-thalassemia major or other congenital hemolytic anemia after splenectomy. A procedure to separate these normoblasts from the other nucleated cells of the peripheral blood was developed, providing us with a convenient source of DNA for investigating parameters related to human erythroid differentiation. Blood samples were obtained from six adult patients who express their gamma-globin genes at different levels. Inverse correlation between methylation and gene activity was consistently observed for five of the eight sites analyzed. A site 3' to the beta gene was always unmethylated, two sites flanking the epsilon gene were always found to be methylated, and two sites 5' to the two gamma genes, G gamma and A gamma, were hypomethylated in correlation with gamma gene activity of the individual patients. A site 5' to the delta gene was unmethylated in normoblasts as well as in WBC. No apparent relation between hypomethylation and gene activity was observed for two additional sites. The results suggest that methylation at specific chromosomal locations participate in genetic regulation of the beta-like globin genes in humans.

Mycobacterium lepraemurium infection of mice produces a chronic lethal disease that is characterized by massive accumulation of macrophages throughout the mononuclear-phagocyte system. We studied the influence of M. lepraemurium infection on the composition and function of the hematopoietic system. Medullary erythropoiesis was virtually abolished, as reflected by a small number of erythroid elements and a decrease in the number and frequency of erythroid progenitors in the bone marrow, together with reduced uptake of 59Fe into bone marrow hemin. On the other hand, erythropoiesis was observed in the spleen, as demonstrated by a large number of erythroid cells, a sixfold increase of 59Fe uptake, and a pronounced increase in the number of erythroid progenitors. A considerable increase of monocyte progenitors was observed in the spleen, and a more modest increase was observed in the bone marrow. This increase may be accounted for, at least in part, by greatly increased levels of macrophage-colony-stimulating factor in the serum of infected mice. Thus, M. lepraemurium infection produces important changes in the hematopoietic system, during the course of which the spleen becomes the major hematopoietic organ.