Search

A culture procedure for growing erythroid progenitors in liquid medium is described. The procedure is divided into two phases. The first is an erythropoietin (EPO)–independent phase in which peripheral blood mononuclear cells are isolated and cultured in the presence of a combination of growth factors, but in the absence of EPO. During this phase, early erythroid‐committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an EPO‐supplemented medium, continue to proliferate and mature into hemoglobin‐containing nucleated erythroid cells. The culture procedure yields populations that are large, relatively pure, and synchronized (in terms of differentiation), and which recapitulate in vivo erythropoiesis. Since the cells are grown in suspension, samples of cells can be withdrawn at any time, without disturbing the cultures, and assayed for various parameters, e.g., morphology, size, number, viability, apoptosis, cell cycle, surface antigens, or gene expression. Several procedures for analyzing the cultured cells with respect to their hemoglobin content during their differentiation are included.

Human erythroid precursors grown in culture possess membrane receptors that bind and internalize acid isoferritin. These receptors are regulated by the iron status of the cell, implying that ferritin iron uptake may represent a normal physiologic pathway. The present studies describe the fate of internalized ferritin, the mechanisms involved in the release of its iron, and the recognition of this iron by the cell. Normal human erythroid precursors were grown in a 2-phase liquid culture that supports the proliferation, differentiation, and maturation of erythroid precursors. At the stage of polychromatic normoblasts, cells were briefly incubated with 59Fe- and/or125I-labeled acid isoferritin and chased. The125I-labeled ferritin protein was rapidly degraded and only 50% of the label remained in intact ferritin protein after 3 to 4 hours. In parallel, 59Fe decreased in ferritin and increased in hemoglobin. Extracellular holoferritin uptake elevated the cellular labile iron pool (LIP) and reduced iron regulatory protein (IRP) activity; this was inhibited by leupeptin or chloroquine. Extracellular apoferritin taken up by the cell functioned as an iron scavenger: it decreased the level of cellular LIP and increased IRP activity. We suggest that the iron from extracellular is metabolized in a similar fashion by developing erythroid cells as is intracellular ferritin. Following its uptake, extracellular ferritin iron is released by proteolytic degradation of the protein shell in an acid compartment. The released iron induces an increase in the cellular LIP and participates in heme synthesis and in intracellular iron regulatory pathways.

Recombinant human erythropoietin (rhEPO) increases fetal hemoglobin synthesis in nonpregnant thalassaemic patients. We used rhEPO in 4 pregnant patients with heterozygous β-thalassemia and anemia to study its effect on erythropoiesis, F cell production, and HbF synthesis. Patients were treated with a combination therapy of rhEPO and iron. The effect on HbF synthesis was assessed by the percentage of F reticulocytes, F cells, and total HbF, erythropietis by reticulocyte count, and hemoglobin measurements and iron status by ferritin levels, transferrin saturation, and percentage of hypochromic red cells. RhEPO caused an increase of F reticulocytes (1.5 to 10.5 fold), F cells (5.0 to 7.7 fold), and HbF (1.4 to 2.2 fold). All patients showed an increase of young, immature reticulocytes and had elevated reticulocytes at the end of therapy. Hemoglobin increased with a range from 0.3 to 1.5 g/dL. Transferrin saturation and ferritin levels were normal at the end of the study. There was an increase of the percentage of hypochromic red cells, indicating functional iron deficiency after rhEPO administration despite supplemental iron. RhEPO stimulates both HbF synthesis and erythropoiesis in pregnant patients with heterozygous β-thalassemia and anemia. Since it is known that high HbF levels ameliorate thalassemia symptoms in nonpregnant patients, use of rhEPO for the treatment of severe anemia in thalassaemic patients during pregnancy might be further evaluated. J. Matern.-Fetal Med. 1999;8:1–7. © 1999 Wiley-Liss, Inc.

Objective: Recombinant human erythropoietin (rhEPO) increases fetal hemoglobin synthesis in nonpregnant thalassaemic patients. We used rhEPO in 4 pregnant patients with heterozygous β-thalassemia and anemia to study its effect on erythropoiesis, F cell production, and HbF synthesis.Methods: Patients were treated with a combination therapy of rhEPO and iron. The effect on HbF synthesis was assessed by the percentage of F reticulocytes, F cells, and total HbF, erythropietis by reticulocyte count, and hemoglobin measurements and iron status by ferritin levels, transferrin saturation, and percentage of hypochromic red cells.Results: RhEPO caused an increase of F reticulocytes (1.5 to 10.5 fold), F cells (5.0 to 7.7 fold), and HbF (1.4 to 2.2 fold). All patients showed an increase of young, immature reticulocytes and had elevated reticulocytes at the end of therapy. Hemoglobin increased with a range from 0.3 to 1.5 g/dL. Transferrin saturation and ferritin levels were normal at the end of the study. There was an increase of the percentage of hypochromic red cells, indicating functional iron deficiency after rhEPO administration despite supplemental iron.Conclusions: RhEPO stimulates both HbF synthesis and erythropoiesis in pregnant patients with heterozygous β-thalassemia and anemia. Since it is known that high HbF levels ameliorate thalassemia symptoms in nonpregnant patients, use of rhEPO for the treatment of severe anemia in thalassaemic patients during pregnancy might be further evaluated.

Human erythroid precursors grown in culture possess membrane receptors that bind and internalize acid isoferritin. These receptors are regulated by the iron status of the cell, implying that ferritin iron uptake may represent a normal physiologic pathway. The present studies describe the fate of internalized ferritin, the mechanisms involved in the release of its iron, and the recognition of this iron by the cell. Normal human erythroid precursors were grown in a 2-phase liquid culture that supports the proliferation, differentiation, and maturation of erythroid precursors. At the stage of polychromatic normoblasts, cells were briefly incubated with 59Fe- and/or125I-labeled acid isoferritin and chased. The125I-labeled ferritin protein was rapidly degraded and only 50% of the label remained in intact ferritin protein after 3 to 4 hours. In parallel, 59Fe decreased in ferritin and increased in hemoglobin. Extracellular holoferritin uptake elevated the cellular labile iron pool (LIP) and reduced iron regulatory protein (IRP) activity; this was inhibited by leupeptin or chloroquine. Extracellular apoferritin taken up by the cell functioned as an iron scavenger: it decreased the level of cellular LIP and increased IRP activity. We suggest that the iron from extracellular is metabolized in a similar fashion by developing erythroid cells as is intracellular ferritin. Following its uptake, extracellular ferritin iron is released by proteolytic degradation of the protein shell in an acid compartment. The released iron induces an increase in the cellular LIP and participates in heme synthesis and in intracellular iron regulatory pathways.

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.

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.

Hereditary pyropoikilocytosis (HPP) is a recessively inherited hemolytic anemia characterized by severe poikilocytosis and red blood cell fragmentation. HPP red blood cells are partially deficient in spectrin and contain a mutant alpha or beta-spectrin that is defective in terms of spectrin self-association. Although the nature of the latter defect has been studied in considerable detail and many mutations of alpha-spectrin and beta spectrin have been identified, the molecular basis of spectrin deficiency is unknown. Here we report two mechanisms underlying spectrin deficiency in HPP. The first mechanism involves a thalassemia-like defect characterized by a reduced synthesis of alpha-spectrin as shown by studies involving synthesis of spectrin in two unrelated HPP probands and their parents: One parent carries the elliptocytogenic spectrin mutation, whereas the other parent is fully asymptomatic. Peripheral blood mononuclear cells as a source of erythroid burst-forming unit (BFUe) were cultured in a two-phase liquid culture system that gives rise to terminally differentiated erythroblasts. Pulse-labeling studies of an equal number of erythroblasts or morphologically identical maturity showed that the synthesis of alpha-spectrin as well as the mRNA levels as measured by the competitive polymerase chain reaction (PCR) method are markedly reduced in the presumed asymptomatic carriers and the HPP probands. In contrast, the synthesis and mRNA levels of beta-spectrin were normal. These results constitute a direct demonstration of an alpha-spectrin synthetic defect in a subset of asymptomatic carriers of HPP and HPP probands. The second mechanism underlying spectrin deficiency involves increased degradation of mutant spectrin before its assembly on the membrane. This is evidenced by pulse labeling studies of erythroblasts from a patient with HPP associated with a homozygous state for spectrin alpha I/46 mutation (leu-pro mutation at AA 207 of alpha-spectrin). These studies showed that although spectrin is synthesized in the cytosol in normal amounts, the rate of turnover of alpha-spectrin is faster resulting in about 40% to 50% reduced assembly of alpha-spectrin and beta-spectrin on the membrane. Thus, spectrin deficiency in this case is at least in part caused by increased susceptibility of the mutant spectrin to degradation before its assembly on the membrane. We conclude that at least two separate mechanisms underlie the molecular basis of spectrin deficiency in HPP.

Iron delivery to mammalian cells is traditionally ascribed to diferric transferrin (Tf). We recently reported that human erythroid precursor cells possess specific membranes receptors that bind and internalize acid isoferritin. Here we show that ferritin uptake by these cells is highly regulated and that the internalized ferritin-iron is used for home synthesis and thus, this process could constitute a physiological pathway for iron assimilation. Ferritin was internalized by a specific, saturable process, distinct from the uptake of iron associated with albumin. Ferritin uptake downregulated transferrin-receptor expression, indicating that internalized ferritin-iron was recognized as an integral part of the cellular iron content. Ferritin receptor expression was coordinated to cell development and was tightly regulated by cellular iron status. Receptor abundance was increased by iron-depletion and decreased by iron-loading, while the affinity of the ferritin receptor for acid isoferritin remained nearly constant (kd = 4.1 +/- 0.5 x 10(-6) mol/L). Under all experimental conditions, ferritin- and transferrin-receptor expression was closely coordinated, suggesting that these pathways possess a common regulatory element. It is concluded that ferritin uptake by erythroid cells constitutes an iron uptake pathway in addition to the classical transferrin uptake pathway.

The effects of heme, when added as the ferric chloride salt, hemin, on human erythroid cells grown in a two-phase liquid culture system were studied. When added together with erythropoietin, on initiation of the second phase of the culture, hemin greatly accelerated hemoglobin (Hb) accumulation in these cells. The effect was greater during their early stages of maturation, suggesting that heme availability is then a rate-limiting step for Hb synthesis. Hemin increased preferentially the production of fetal Hb (HbF) compared with adult Hb; this was associated with a selective twofold elevation in gamma-mRNA levels. Using succinylacetone, a potent inhibitor of heme synthesis, we showed that exogenously supplied hemin could be incorporated into the de novo formed Hb. Therefore, the mechanism of hemin action may be several fold, including effects on globin gene transcription and posttranslational events, eg, supplying the prosthetic group for Hb assembly. Hemin increased HbF of cells derived from patients with sickle cell anemia and beta-thalassemia as well as that of cells from normal donors. Moreover, when added in combination with other HbF-augmenting agents such as the cytotoxic drug, hydroxyurea, a synergistic effect was obtained, with considerably less cytotoxicity than with hydroxyurea alone. These results have clinical potential in light of the ameliorating effect that increased HbF has in patients with genetic diseases of the beta-globin chain and raise the possibility of combined treatment with hemin and other drugs now being used to treat these diseases.

Abstract: Peripheral blood samples from 61 patients (36 male, 25 female) with all stages of B-type chronic lymphocytic leukemia (CLL) were studied for MDR1 phenotype using monoclonal antibodies and rhodamine-123 dye exclusion, a functional assay of MDR1 expression. The duration of the disease varied from 1 month to 22 years at the time of initial study. Overall, 74% of the patients were positive for rhodamine-123 exclusion. When analyzed by gender, significantly more men than women were positive (89% versus 48%, p<0.001). There were more positive men than women for every stage of the disease. Female patients were found to be either MDR1 phenotype positive or negative at any stage of the disease. In contrast, all male patients with early (stages 0–II) disease were MDR1 phenotype positive. One early-stage (stage II) male patient converted from rhodamine-efflux positive to rhodamine-efflux negative as he progressed from stage-II to stage-IV disease. We suggest that some of the differences in disease biology of male versus female CLL patients (women having a more benign course) may be due to gender-dependent differences in drug-resistance gene activity, including MDR1. Our results also emphasize the need to take into account gender in evaluating the clinical course of patients with CLL.

The aim of this study was to diagnose lipid storage diseases in embryos at the preimplantation stage. Two parallel approaches were employed. Firstly, activities of several sphingolipid hydrolases were determined in extracts of murine embryos and also human oocytes and polyspermic embryos. Sensitive fluorescent or fluorogenic procedures provided indications that Tay-Sachs, Gaucher and Krabbe diseases might be diagnosed in one human blastomere, while for Niemann-Pick disease two might be required. Secondly, pyrene lipids were administered into murine embryos and their fluorescence was quantified by computerized imaging microscopy. As a model of Gaucher disease, the fluorescent substrate pyrene glucosylceramide was administered into murine embryos in the presence or absence of an inhibitor of the enzyme beta-glucosidase. Because of decreased degradation of the substrate in enzyme-inhibited cells, the fluorescence per blastomere was considerably greater relative to those which received no inhibitor. The results indicated that lipid storage diseases might be diagnosed in single human blastomeres at the preimplantation stage, obviating the need for pre-natal diagnosis and abortion of affected foetuses.

Hemoglobin and ferritin iron content have been followed during differentiation in tissue cultures of murine erythroleukemia cells (MELC) using the techniques of Mössbauer spectroscopy and electron microscopy. In undifferentiated cells grown without DMSO, only iron stored in ferritin was detected. The amount of iron in a cell grown in the presence of iron citrate is ~1.2 × 10–14g, whereas in a cell grown in the presence of transferrin the amount is ~ 0.28 × 10–14g. These quantities do not depend on the iron concentration in the nutrition medium in a range from 0.3 to 2.0 µgFe/ml and are the same for growth times between 8 hr and 7 days. Cells grown with DMSO contain, in addition to ferritin, increasing concentrations of hemoglobin. Chase experiments prove that ferritin iron participates in hemoglobin synthesis. The amount of ferritin iron reaches saturation within less than 8 hr in MELC grown with or without DMSO. In differentiating cells grown with iron citrate there is a decrease with time in ferritin iron content concomitant with the increase in hemoglobin. Cells grown with transferrin incorporate additional amounts of iron, which are approximately equal to the amounts used for hemoglobin synthesis maintaining a constant ferritin iron level. In the electron microscope, iron is seen only as ferritin within lysosomes. The density of the ferritin in lysosomes correlates with the ferritin iron concentrations determined by Mossbauer spectroscopy.

The use of chromosome banding techniques has provided a valuable diagnostic tool in various malignancies. The application of these methods, however, is often restricted by a low yield of mitotic cells and the patient's unwillingness to comply with repeated bone marrow aspiration. In an attempt to promote mitotic activity of leukemic cells from the bone marrow and peripheral blood, we employed a new method based on culturing the cells in the presence of a conditioned medium derived from a human bladder carcinoma cell line (5637). In addition to colony stimulating factor, this conditioned medium contains a factor that is capable of stimulating leukemic myeloblast proliferation. Bone marrow and peripheral blood mononuclear cells from 58 patients with a variety of myeloid leukemias were cultured for 24 to 120 hours in the presence or absence of conditioned medium. These bone marrow cells showed a pronounced increase in the mitotic index (5- to 50-fold) as compared to unstimulated cultures, and a greater than 100-fold increase as compared to fresh, uncultured bone marrow cells. Analyzable metaphases could be obtained even in marrow samples in which direct or 24-hour G-banding techniques had failed to reveal metaphases. The effect observed on peripheral blood cells was even more dramatic because prior to culture no mitotic cells were detected, whereas up to 2% mitotic cells were found in conditioned medium-stimulated peripheral leukemic cells. Karyotype analysis of 36 out of the 58 leukemic patients has shown that the chromosome changes discovered in conditioned medium stimulated cells were identical to those found in unstimulated cells. New chromosome aberrations, attributable to the stimulation of growth by conditioned medium, were not found. The quality of the metaphases analyzed following conditioned medium stimulation was considerably better than that of unstimulated samples. Frozen cells, when cultured with conditioned medium, were also suitable for cytogenetic analysis. Thus, the use of this conditioned medium permits adequate cytogenetic analysis even in cases where such analysis was previously impossible.

Hemoglobin and ferritin iron content have been followed during differentiation in tissue cultures of murine erythroleukemia cells (MELC) using the techniques of Mossbauer spectroscopy and electron microscopy. In undifferentiated cells grown without DMSO, only iron stored in ferritin was detected. The amount of iron in a cell grown in the presence of iron citrate is approximately 1.2 X 10(-14) g, whereas in a cell grown in the presence of transferrin the amount is approximately 0.28 X 10(-14) g. These quantities do not depend on the iron concentration in the nutrition medium in a range from 0.3 to 2.0 microgram Fe/ml and are the same for growth times between 8 hr and 7 days. Cells grown with DMSO contain, in addition to ferritin, increasing concentrations of hemoglobin. Chase experiments prove that ferritin iron participates in hemoglobin synthesis. The amount of ferritin iron reaches saturation within less than 8 hr in MELC grown with or without DMSO. In differentiating cells grown with iron citrate there is a decrease with time in ferritin iron content concomitant with the increase in hemoglobin. Cells grown with transferrin incorporate additional amounts of iron, which are approximately equal to the amounts used for hemoglobin synthesis maintaining a constant ferritin iron level. In the electron microscope, iron is seen only as ferritin within lysosomes. The density of the ferritin in lysosomes correlates with the ferritin iron concentrations determined by Mossbauer spectroscopy.

The present paper reports on the application of a simple micromethod for quantitation of total and fetal hemoglobin in small volumes of diluted erythroid cell hemolysates. A modified alkaline denaturation procedure was used to denature and precipitate non-fetal hemoglobins and the tetramethylbenzidine procedure was used for quantitation of the soluble hemoglobin. Using a combination of these procedures, total hemoglobin concentrations as low as 0.2 mg% (2 μg/ml) and a fetal hemoglobin proportion of 0.5% could be reliably determined. We have utilized this procedure to study two aspects of hemoglobin production in human erythroid cells grown in culture; the proportion of fetal hemoglobin of cells in culture as compared to that in the peripheral blood of the donors, and the kinetics of hemoglobin production during maturation of these cultured cells. The results show that the cultured erythroid cells have a hemoglobin phenotype similar to the individual from whom they are derived and that erythroid differentiation may be studied in vim in these cultured cells by measurement of these and other parameters.

Plasminogen activator (PA) activity of human promyelocytic leukemia cell line HL‐60 was assayed by following the conversion of plasminogen to plasmin and the plasmin‐mediated hydrolysis of 14C‐labeled globin. When HL‐60 cells were induced to differentiate into macrophages by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), cell‐associated PA activity and secretion of PA into the conditioned medium increased profoundly. PA activity increased earlier and as a result of lower concentrations of TPA than the ability of the cells to adhere. Exposure to 10−6M dexamethasone did not prevent TPA‐induced adherence and produced a slight inhibition of cellular PA activity. These findings imply that TPA‐induced differentiation of HL‐60 cells to macrophage‐like cells is associated with induction of PA activity.

Mössbauer studies in murine (MEL) and human K-562 erythroleukemia cell lines have been utilized to study the fate of iron during intracellular Hb synthesis and denaturation. The results showed that ferritin can serve as an intermediate iron pool for Hb synthesis and for storage of iron released during intracellular Hb denaturation.

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.

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.

Plasminogen activator (PA) activity of human promyelocytic leukemia cell line HL-60 was assayed by following the conversion of plasminogen to plasmin and the plasmin-mediated hydrolysis of 14C-labeled globin. When HL-60 cells were induced to differentiate into macrophages by 12- O-tetradecanoyl-phorbol-13-acetate (TPA), cell-associated PA activity and secretion of PA into the conditioned medium increased profoundly. PA activity increased earlier and as a result of lower concentrations of TPA than the ability of the cells to adhere. Exposure to 10 −6M dexamethasone did not prevent TPA-induced adherence and produced a slight inhibition of cellular PA activity. These findings imply that TPA-induced differentiation of HL-60 cells to macrophage-like cells is associated with induction of PA activity.

Previous studies demonstrated that 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a tumor promoter, is a potent inhibitor of inducer-mediated differentiation of murine erythroleukemia cells. Inhibition of cell differentiation was associated with inhibition of cell growth. The present studies, employing a cell line adapted for growth in TPA, demonstrate that inhibition of differentiation is not dependent upon inhibition of cell growth or a change in the cell division cycle; neither is inhibition of differentiation accompanied by detectable effect on cell uptake of [3H]hexamethylene bisacetamide, the inducer used in these studies. TPA causes an inhibition of expression of all hexamethylene bisacetamide-inducible erythroid characteristics measured, including commitment to terminal cell division, accumulation of globin mRNA, and synthesis of globins, spectrin, heme synthetic enzymes (delta-aminolevulinic acid dehydratase and uroporphyrinogen-I synthase) and heme. A hypothetical model for the inhibitory action of tumor promoters on terminal cell differentiation is discussed.

The diterpene tumor promoters enhance the proliferation in culture of myeloid and erythroid precursor cells from normal mouse hemopoietic tissues. The effect is observed only with those diterpenes that are tumor promoters, including 12-O-tetradecanoylphorbol 13-acetate (TPA); diterpenes that are inactive as tumor promoters are ineffective as stimulators of colony formation. Tumor promoters act synergistically with suboptimal concentrations of conditioned medium used as a source of colony-stimulating factor (CSF) to increase both the number and size of myeloid colonies. Formation of myeloid colonies is stimulated by tumor promoters even without addition of CSF. Both pure and mixed granulocyte/macrophage colonies develop; high concentrations (> 100 micrograms/ml) of TPA are more favorable for macrophage colony formation. In erythropoietin-stimulated cultures, tumor promoters enhance the development of relatively early and intermediate of erythroid precursors, whereas later erythroid precursors are unaffected.

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.

Hydroxyurea (HU), an inhibitor of DNA synthesis, has been shown to increase fetal hemoglobin (HbF) levels in patients with sickle cell anemia and in some patients with beta-thalassemia. However, until now there have not been good in vitro model systems that simulate this effect for study of the molecular and cellular mechanism(s) involved in perturbing the normal ontogeny of the globin genes. We analyzed the cellular effects of HU using a two-phase liquid culture procedure (Fibach et al: Blood 73:100, 1989) in which human peripheral blood- derived progenitor cells undergo proliferation and differentiation. HU was found to have multiple effects on these cultured cells: (1) an increase in the proportion of HbF produced; (2) a decrease in cell number due to inhibition of cell proliferation; (3) an increase in hemoglobin content per cell (mean corpuscular hemoglobin [MCH]); and (4) an increase in cell size (mean corpuscular volume). The extent of these effects was related to the HU dose and time of addition. When added to cell cultures from normal individuals, 4 days following their exposure to erythropoietin (EPO), 100 mumol/L HU caused a 1.3- to 3.5- fold increase in the proportion of HbF, from 0.4% to 5.2% (mean 1.6) in untreated to 1.5% to 8.2% (mean 3.1) in HU-treated cultures and a 45% +/- 10% increase in MCH but only a 25% +/- 7% decrease in cell number on day 13. Cultures of cells derived from five patients with sickle cell anemia have shown a twofold to fivefold increase in the percentage of Hb F following addition of HU while four patients with beta- thalassemia showed a 1.3- to 6.2-fold increase. We believe that this primary cell culture procedure should prove useful in studying the cellular and molecular mechanisms of pharmacologic induction of HbF and might provide a valuable predictive assay system for evaluation of the response of individual patients with hemoglobinopathies to HU and similar agents.

Aqueous dispersions of 12-(1-pyrene)-dodecanoic acid (P12), a medium-chain fatty acid to which the fluorescent probe pyrene has been covalently linked, shows a considerable increase in fluorescence when the probe is introduced into a hydrophobic environment. This enables the uptake of P12 by liposomes and cells to be followed directly in a spectrofluorometer, without separating the cells from the P12-containing medium. In the present study, we show that complexing P12 to albumin produced a very high fluorescence emission intensity. This made direct measurements of the uptake by cells of albumin-bound P12 impossible. Such direct measurements could, however, be made using albumin which had been interacted with trinitrobenzenesulphonic acid (TNBS). The yellow trinitrophenyl (TNP) residues, which were thereby covalently linked to the albumin, quenched the fluorescence of pyrene in the TNP-albumin/P12 complex. Upon release of the P12 molecules from this complex and their subsequent uptake by cells, fluorescence increased. This technique was utilized for the continuous monitoring of the uptake of P12 by different cell types and cells at various stages of maturation.

Introduction of DNA into human hemopoietic cells is required for the study of regulatory mechanisms operating in these cells, as well as for possible procedures of gene therapy. However, with hemopoietic cells the conventional technique of calcium phosphate precipitation is inefficient. The pathway of encapsidation of plasmid DNA as simian virus 40 (SV40) pseudovirions for the introduction of new genetic material was therefore investigated. Encapsidation was achieved in COS (monkey kidney) cells, which express SV40 large tumor (T) antigen constitutively. The vector, pSO, was introduced to the COS cells by DNA transfection. It carried the SV40 origin of replication (ori), to facilitate replication of the plasmid in the COS cells. The SV40 capsid proteins were supplied in trans by a helper SV40 virus. The bacterial chloramphenicol acetyltransferase gene cat was used as a model for gene transmission. After encapsidation, the pseudovirions were used in infection of the human erythroleukemic cell line K562 and of normal human bone marrow cells. The results demonstrate that the cat gene can be transmitted with high efficiency. Over 40% of the infected K562 cells and 30% of the infected bone marrow cells were observed to contain plasmid DNA 48 hr after infection. Moreover, the results suggest that the efficiency of gene transmission by this vector can be improved and so may approach the theoretical 100%.

The developmental changes in hemoglobin gene expression known as "switching" involve both the sequential activation and silencing of the individual globin genes. We postulated that in addition to changes in transcription, posttranscriptional mechanisms may be involved in modulating globin gene expression. We studied globin RNA transcripts in human adult erythroid cells (hAEC to analyze the mechanism of silencing of the embryonic epsilon-globin gene in the adult stage and in K562 erythroleukemic cells to analyze the inactive state of their adult beta-globin genes. In hAEC, which express primarily the beta-globin gene, quantitative PCR analysis shows that beta-mRNA exon levels are high and comparable among the three exons; the RNA transcripts corresponding to exons of the gamma-globin gene are low, with slight differences among the three exons. Although epsilon-globin is not expressed, epsilon-globin RNA transcripts are detected, with exon I levels comparable to that of gamma-globin exon I and much higher than epsilon-exons II and III. As expected, in K562 cells that express high levels of epsilon- and gamma-globin, epsilon- and gamma-mRNA levels are high, with comparable levels of exons I, II, and III. In K562 cells beta-mRNA levels are very low but beta-exon I levels are much higher than that of exons II or III. Moreover, all or most of the globin transcripts for the highly expressed globin genes in both cell types (gamma and beta in hAEC, epsilon and gamma in K562 cells) found in the cytoplasm or nucleus are correctly processed. The globin transcripts that are detected both in the cytoplasm and nucleus of cells without expression of the corresponding protein are largely unspliced (containing one or two intervening sequences). These studies suggest that in addition to changes in transcription rates, changes in completion or processing of globin RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype.

Addition of the potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), to murine erythroleukemia cell lines in suspension cultures inhibited both spontaneous differentiation and differentiation induced by hexamethylene bisacetamide (HMBA), dimethyl sulfoxide, or butyric acid. Inhibition was unrelated to cytotoxicity and was reversible. When several plant diterpenes were tested, there was a positive correlation between tumor-promoting activity and inhibition of differentiation. TPA inhibited HMBA-induced differentiation only if added prior to the time of commitment to differentiation, as assayed by scoring for differentiation after transfer of cells from HMBA to fresh medium without HMBA. TPA-mediated inhibition of differentiation was associated with a decrease in globin mRNA accumulation.

This report describes in detail the procedures for growing human erythroid cells in liquid culture for evaluating the potential of pharmacological agents to affect hemoglobin production. The procedure consists of two phases: an erythropoietin-independent phase in which peripheral blood mononuclear cells are first cultured in the presence of a combination of hemopoietic growth factors, but in the absence of erythropoietin, where early erythroid committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an erythropoietin-supplemented medium, continue to proliferate and mature into orthochromatic normoblasts and enucleated erythrocytes. This procedure produces large cultures of relatively pure and synchronized erythroid cell populations derived from normal donors or patients with β hemoglobinopathies. The cultured cells recapitulate many aspects of erythropoiesis in vivo, including the donor's pattern of hemoglobin production (types and proportions). Tested compounds, at different concentrations, are added at different stages of the culture. The various types of hemoglobins and globin chains produced can be measured by high performance liquid chromatographic techniques and their cellular distribution analyzed by flow cytometry using fluorescently labeled antibodies against specific hemoglobins. This approach provides a screening system for compounds with potential therapeutic efficacy in patients with β hemoglobinopathies.

Three flow cytometric methods of counting F cells were evaluated in the settings of an external laboratory assessment scheme. The laboratories to participate with a different method were located in Oxford (method O), Athens (method A) and Jerusalem, (method J). Two monoclonal anti-γ chain antibodies were used: monoclonal antibody produced by P. Beverley (Oxford) (BEV) and an antibody provided by Bioatlantic S.A.R.L. (France) (BIO). The specimens tested were mixtures in five predefined ratios of a sample with homozygous δβ-thalassemia with 100% F cells with a sample with no F cells. A central independent laboratory prepared and distributed the aliquots (at room temperature) to the participating centers within 2 (O), 3 (A), and 6 (J) days. The performance of the three methods was evaluated by: 1) deviation indices, 2) relative accuracy, as percent difference of the counts from the target values, and 3) bias and linearity by linear regression of the counts versus the target values [parameters: slope (s), y-intercept (y), R squared (Rs), and F ratio]. The highest score of performance was obtained by method A with both monoclonal antibodies.

Binding of growth hormone (GH) and erythropoietin (EPO) to their respective receptors results in receptor clustering and activation of tyrosine kinases that initiate a cascade of events resulting not only in the rapid tyrosine phosphorylation of several proteins but also in the induction of early-response genes. In this report, we show that GH and EPO induce the tyrosine phosphorylation of cellular proteins with molecular masses of 93 kDa and of 91 and 84 kDa, respectively, and that these proteins form DNA-binding complexes which recognize an enhancer that has features in common with several rapidly induced genes such as c-fos. Assembly of the protein complexes required tyrosine phosphorylation, which occurred within minutes after addition of ligand. The activated complexes translocated from the cytoplasm to the nucleus. The protein activated by GH is antigenically similar to p91, a protein common to several transcription complexes that are activated by interferons and other cytokines. In contrast, the proteins activated by EPO are distinct from p91. These findings establish the outlines for a cytokine-induced intracellular signaling pathway, which begins with ligand-induced receptor clustering that activates one or more tyrosine kinases. These data are the first to demonstrate that GH- and EPO-activated tyrosine-phosphorylated proteins can specifically recognize a well-defined enhancer and therefore provide a mechanism for rapidly transducing signals from the membrane to the nucleus.

Murine erythroleukemia (MEL) and human K-562 cell lines were cultured in the presence of 57Fe, and the quantities of cellular iron-containing compounds were determined with the aid of Mossbauer spectroscopy. Upon induction of differentiation, both ferritin-iron and hemoglobin (Hb) iron could be detected. Treatment of the cells with 0.01%-0.02% acetylphenylhydrazine (APH) resulted in gradual denaturation of Hb and incorporation of the released Hb-iron into ferritin. Following treatment with APH, the ratio of Hb-57Fe to ferritin-57Fe decreased from 2.6 to 0.2 in MEL cells and from 0.56 to 0.12 in K-562 cells. No change was observed in the total intracellular iron. Using fluorescence ELISA, an increased level of immunologically detectable ferritin was found in hemoglobinized K-562 cells treated with APH, as compared to the amount of ferritin found in untreated cells. Ferritin may thus function not only as an intermediate during Hb synthesis, but also as storage protein for iron released during Hb denaturation.

Murine erythroleukemia (MEL) and human K-562 cell lines were cultured in the presence of 67Fe, and the quantities of cellular iron-containing compounds were determined with the aid of Mössbauer spectroscopy. Upon induction of differentiation, both ferritin-iron and hemoglobin (Hb) iron could be detected. Treatment of the cells with 0.01%-0.02% acetylphenylhydrazine (APH) resulted in gradual denaturation of Hb and incorporation of the released Hb-iron into ferritin. Following treatment with APH, the ratio of Hb-67Fe to ferritin-67Fe decreased from 2.6 to 0.2 in MEL cells and from 0.56 to 0.12 in K-562 cells. No change was observed in the total intracellular iron. Using fluorescence ELISA, an increased level of immunologically detectable ferritin was found in hemoglobinized K-562 cells treated with APH, as compared to the amount of ferritin found in untreated cells. Ferritin may thus function not only as an intermediate during Hb synthesis, but also as storage protein for iron released during Hb denaturation.

Vesicular stomatitis virus (VSV) was incubated with pyrene dodecanoic acid (P12), a fluorescent derivative of a medium-chain length fatty acid, and subjected to irradiation with a long wavelength ultra-violet light source at 366 nm (UVA). This inactivated the virus, resulting in a drastic decrease of its infectivity. The virus inactivation was proportional to the concentration of the pyrene-fatty acid, the length of exposure of the virus to P12 and the irradiation dose.

Vesicular stomatitis virus (VSV) was incubated with pyrene dodecanoic acid (P12), a fluorescent derivative of a medium‐chain length fatty acid, and subjected to irradiation with a long wavelength ultra‐violet light source at 366 nm (UVA). This inactivated the virus, resulting in a drastic decrease of its infectivity. The virus inactivation was proportional to the concentration of the pyrene‐fatty acid, the length of exposure of the virus to P12 and the irradiation dose.

Hydroxyurea (HU) is a widely used cytotoxic agent that is known to induce fetal hemoglobin (HbF) production. HU-induced HbF production is beneficial for some patients with β-thalassemia and also ameliorates the severity of pain episodes in sickle cell anemia. Various cell culture systems have been used to elucidate the implicated mechanisms. We have previously demonstrated in both K562 human erythroleukemic cells and human erythroid cells (hAEC) that HU increases the transcription of low-expressed globin genes as well as the splicing efficiency of primary globin transcripts; however, other post-transcriptional effects could not be excluded. In this study, we extended our analysis in order to further understand the effect of HU on erythropoiesis and the molecular mechanisms involved in globin gene elevation. Peripheral blood hAEC from normal individuals were cultured in a two-phase liquid culture. Erythropoietin was added only to phase II. Erythroid cells in phase II were exposed to different HU concentrations 25/50/100/300 μM at day 5 (proerythroblast stage) either continuously (1– 3 days) or were pulse treated [initially exposed to HU (1–3 days), then washed and harvested at day 8–14)]. Hb-containing cell number, measured by flow cytometry (FC), was affected by continuous as well as pulse HU treatment in a dose- and-time dependent fashion. HbF-cell numbers were determined from day 0 of phase II throughout phase II culture by both FC and the immunocytochemical StreptABComplex/AP staining procedure. At day 0 of phase II no HbF cells were detected by either method; at day 8 of phase II, F-cell numbers increased 2-fold in cultures exposed to 100 μM/300 μM HU for 3 days. Hbs and globin chains were separated and measured by ion exchange and reverse-phase HPLC, respectively. HU (300 μ M) treatment caused a 1.5-fold increase in γ-globin chain production and preferentially elevated the Gγ-chain. Quantification of γ-mRNA was performed by real-time PCR. While addition of low HU concentrations (25 and 50 μM) for 1–3 days had no effect, treatment with high concentrations (100/300 μM) caused a significant time- and dose-dependent increase in γ-mRNA (100 μM HU, 1– 3 days: 1.2 to 2.1-fold; 300 μM, 1–3 days: 1.5 to 2.2-fold). Pulse treatment with 100 μM HU (cells exposed for 1 day, washed and harvested 2 days later) led to a greater increase in γ-mRNA levels (2.2-fold) than continuous exposure for 3 days (1.6-fold). HU effects on γ-mRNA stability were analyzed in hAEC cultures-treated with HU and actinomycin-D (actD: 5μg/ml) for 1hr to 24hrs. HU-treated (100 μM) cells exhibited a 3.9-fold increase in γ-mRNA 24 hrs post-actD addition. Exposure at higher HU concentrations and/or prolongation of actD treatment had a toxic effect (decrease in both γ-mRNA and cell numbers). In conclusion, the present study provides evidence that HU can stimulate erythropoiesis and increase Hb production in a dose- and-time dependent fashion; HU effects are mediated by transcriptional and post-transcriptional modulation of γ-globin gene expression.

Ex-vivo expansion strategies of cord blood (CB) derived human progenitor cells (HPC) have been developed to provide an answer to the delayed time to engraftment and to the extended periods of neutropenia and thrombocytopenia encountered. These problems occur in transplants of CB products performed in adults due to the low yield of HPC. Reports correlating the clinical outcome with the number of CD34+cells suggest that the transplantation of ex vivo expanded CD34+cells may shorten the time to engraftment. The use of copper chelators such as tetraethylenepentamine (TEPA) has been shown to prolong expansion of HPC by inhibiting cell differentiation and thus allowing self-renewal of primitive HPC (Exp Hematol. 2004; 32:547). The variability observed in the expansion results, caused by the intrinsic differences among the various sources of CB units and processing methodologies, complicates the interpretation of published results. In the present report we summarize our results of CD34+cell ex-vivo expansion of over 100 units in the presence of IL-6, TPO, Flt-3 ligand and SCF with and without TEPA. After 3 weeks, the total nuclear cell (TNC), colony forming unit (CFU), and the total CD34+cell fold expansion of TEPA-treated cultures were 424±10.5 (n=230), 104±7 (n=112) and 19±3.2 (n=113), respectively, with no significant differences compared to controls. However, the percentage of the primitive subset of HPC, CD34+/38−cells, significantly (p<0.0001) increased in the TEPA-treated cultures (3.2%±0.2, n=59) vs. controls (1.6%±0.27, n=147). In contrast, after 5 weeks in culture, the TNC fold expansion was significantly (p<0.05) higher in TEPA-treated cultures compared to the controls, 1471±63.5 (n=89) vs. 1270 ±240 (n=55), respectively. The increase in TNC in TEPA-treated cultures did not result in increased HPC differentiation, but was accompanied by an increased self-renew capacity of CD34+ cells as represented by a 57±5.9 fold (n=47) vs. a 32±3.5-fold (n=38) amplification in the controls (p<0.0009). The overall fold expansion in culture analyzed by a Kaplan-Meier survival curve function demonstrate that the TNC, CFU, CD34+and CD34+/38−cells derived from TEPA-treated cultures have higher in vitro survival probabilities than controls (p<0.0014). Cumulative values of all parameters were calculated and a transformation performed using the rank procedure. The results underline that TEPA increases CFU potential and CD34+and CD34+/38−content during the ex-vivo expansion (p<0.01). The TEPA supplemented expansion technology was further tested after up-scaling of the processing and culturing procedures. AC133+cells isolated by the CliniMACS device from frozen CB units obtained from 6 different banks. The expansion results of TNC, CD34+ cells, CFU and %CD34/38- were 337±23 (n=57), 21±4.3 (n=19), 133±27.5 (n=19) and 2.7% ±0.7 (n=19) fold, respectively. A clinical trial with TEPA expanded cultures for treatment of leukemia patients is currently ongoing at MD Anderson Cancer Center, USA.

Hydroxyurea (HU) is a widely used cytotoxic agent that is known to induce fetal hemoglobin (HbF) production. HU-induced HbF production is beneficial for some patients with β-thalassemia and also ameliorates the severity of pain episodes in sickle cell anemia. Various cell culture systems have been used to elucidate the implicated mechanisms. We have previously demonstrated in both K562 human erythroleukemic cells and human erythroid cells (hAEC) that HU increases the transcription of low-expressed globin genes as well as the splicing efficiency of primary globin transcripts; however, other post-transcriptional effects could not be excluded. In this study, we extended our analysis in order to further understand the effect of HU on erythropoiesis and the molecular mechanisms involved in globin gene elevation. Peripheral blood hAEC from normal individuals were cultured in a two-phase liquid culture. Erythropoietin was added only to phase II. Erythroid cells in phase II were exposed to different HU concentrations 25/50/100/300 μM at day 5 (proerythroblast stage) either continuously (1– 3 days) or were pulse treated [initially exposed to HU (1–3 days), then washed and harvested at day 8–14)]. Hb-containing cell number, measured by flow cytometry (FC), was affected by continuous as well as pulse HU treatment in a dose- and-time dependent fashion. HbF-cell numbers were determined from day 0 of phase II throughout phase II culture by both FC and the immunocytochemical StreptABComplex/AP staining procedure. At day 0 of phase II no HbF cells were detected by either method; at day 8 of phase II, F-cell numbers increased 2-fold in cultures exposed to 100 μM/300 μM HU for 3 days. Hbs and globin chains were separated and measured by ion exchange and reverse-phase HPLC, respectively. HU (300 μ M) treatment caused a 1.5-fold increase in γ-globin chain production and preferentially elevated the Gγ-chain. Quantification of γ-mRNA was performed by real-time PCR. While addition of low HU concentrations (25 and 50 μM) for 1–3 days had no effect, treatment with high concentrations (100/300 μM) caused a significant time- and dose-dependent increase in γ-mRNA (100 μM HU, 1– 3 days: 1.2 to 2.1-fold; 300 μM, 1–3 days: 1.5 to 2.2-fold). Pulse treatment with 100 μM HU (cells exposed for 1 day, washed and harvested 2 days later) led to a greater increase in γ-mRNA levels (2.2-fold) than continuous exposure for 3 days (1.6-fold). HU effects on γ-mRNA stability were analyzed in hAEC cultures-treated with HU and actinomycin-D (actD: 5μg/ml) for 1hr to 24hrs. HU-treated (100 μM) cells exhibited a 3.9-fold increase in γ-mRNA 24 hrs post-actD addition. Exposure at higher HU concentrations and/or prolongation of actD treatment had a toxic effect (decrease in both γ-mRNA and cell numbers). In conclusion, the present study provides evidence that HU can stimulate erythropoiesis and increase Hb production in a dose- and-time dependent fashion; HU effects are mediated by transcriptional and post-transcriptional modulation of γ-globin gene expression.

Ex-vivo expansion strategies of cord blood (CB) derived human progenitor cells (HPC) have been developed to provide an answer to the delayed time to engraftment and to the extended periods of neutropenia and thrombocytopenia encountered. These problems occur in transplants of CB products performed in adults due to the low yield of HPC. Reports correlating the clinical outcome with the number of CD34+ cells suggest that the transplantation of ex vivo expanded CD34+ cells may shorten the time to engraftment. The use of copper chelators such as tetraethylenepentamine (TEPA) has been shown to prolong expansion of HPC by inhibiting cell differentiation and thus allowing self-renewal of primitive HPC (Exp Hematol.2004;32:547). The variability observed in the expansion results, caused by the intrinsic differences among the various sources of CB units and processing methodologies, complicates the interpretation of published results. In the present report we summarize our results of CD34+ cell ex-vivo expansion of over 100 units in the presence of IL-6, TPO, Flt-3 ligand and SCF with and without TEPA. After 3 weeks, the total nuclear cell (TNC), colony forming unit (CFU), and the total CD34+ cell fold expansion of TEPA-treated cultures were 424±10.5 (n=230), 104±7 (n=112) and 19±3.2 (n=113), respectively, with no significant differences compared to controls. However, the percentage of the primitive subset of HPC, CD34+/38− cells, significantly (p<0.0001) increased in the TEPA-treated cultures (3.2%±0.2, n=59) vs. controls (1.6%±0.27, n=147). In contrast, after 5 weeks in culture, the TNC fold expansion was significantly (p<0.05) higher in TEPA-treated cultures compared to the controls, 1471±63.5 (n=89) vs. 1270 ±240 (n=55), respectively. The increase in TNC in TEPA-treated cultures did not result in increased HPC differentiation, but was accompanied by an increased self-renew capacity of CD34+ cells as represented by a 57±5.9 fold (n=47) vs. a 32±3.5-fold (n=38) amplification in the controls (p<0.0009). The overall fold expansion in culture analyzed by a Kaplan-Meier survival curve function demonstrate that the TNC, CFU, CD34+ and CD34+/38− cells derived from TEPA-treated cultures have higher in vitro survival probabilities than controls (p<0.0014). Cumulative values of all parameters were calculated and a transformation performed using the rank procedure. The results underline that TEPA increases CFU potential and CD34+ and CD34+/38− content during the ex-vivo expansion (p<0.01). The TEPA supplemented expansion technology was further tested after up-scaling of the processing and culturing procedures. AC133+ cells isolated by the CliniMACS device from frozen CB units obtained from 6 different banks. The expansion results of TNC, CD34+ cells, CFU and %CD34/38- were 337±23 (n=57), 21±4.3 (n=19), 133±27.5 (n=19) and 2.7% ±0.7 (n=19) fold, respectively. A clinical trial with TEPA expanded cultures for treatment of leukemia patients is currently ongoing at MD Anderson Cancer Center, USA.