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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.

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.

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.

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.

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.

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.

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%.

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.