The effect of thrombopoietin on erythroid progenitors in Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare congenital red blood cell aplasia. Usually, erythropoiesis in vitro is defective, and decreased numbers of erythroid progenitors are found in colony assays performed with bone marrow cells. Recently, some investigators showed that thrombopoietin (TPO) also works on common multipotent progenitor cells and stimulates erythropoiesis. We examined the effect of TPO together with erythropoietin (EPO), stem cell factor (SCF), and/or interleukin-3 (IL-3) on erythroid burst-forming units (BFU-E) of bone marrow nonadherent mononuclear cells from 2 patients with DBA using a serum-free culture system. Very few BFU-E appeared in cultures containing EPO alone. Adding IL-3, SCF, or both to cultures containing EPO induced an increase in the number of BFU-E. When TPO was added to cultures containing EPO and IL-3, SCF, or both, the number of BFU-E further increased. Especially in patient 2, BFU-E formation was grossly equal to that in normal controls. We conclude that TPO enhances bone marrow erythropoiesis in patients with DBA in the presence of EPO and SCF and/or IL-3. The data raise the possibility of the combination of TPO and SCF as a therapeutic agent in DBA.     

Developmental regulation of erythropoiesis by hematopoietic growth factors: analysis on populations of BFU-E from bone marrow, peripheral blood, and fetal liver

Fetal hematopoiesis is characterized by expanding erythropoiesis to support a continuously increasing RBC mass. To explore the basis for this anabolic, nonhomeostatic erythropoiesis, the proliferative effect of recombinant hematopoietic growth factors on highly enriched hematopoietic progenitor cells from fetal and adult tissues were compared. Fetal hepatic BFU-E, unlike adult bone marrow (BM) or peripheral blood (PB) BFU-E, were capable of proliferating in response to erythropoietin in the absence of added GM colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), and erythropoietin (Epo) directly stimulated the expansion of the fetal BFU-E pool in suspension culture. A murine monoclonal antibody (MoAb), Ep 3, was raised against enriched fetal liver progenitor cells, which detected all fetal BFU-E and which reacted with the erythropoietin-responsive, GM-CSF/IL-3-independent fraction of adult BM BFU-E and CFU-E. All adult PB BFU-E were Ep 3- but became Ep 3+ after stimulation with GM-CSF or IL-3. These data indicate that Epo plays a unique role in fetal hepatic erythropoiesis, stimulating proliferation of immature BFU-E in addition to promoting terminal differentiation of later erythroid progenitor cells. In addition, these results demonstrate a MoAb which detects all erythropoietin-responsive progenitor cells and distinguishes the BFU-E compartments in adult BM and PB.     

In vitro effects of high titers of recombinant human erythropoietin on the bone marrow erythroid progenitors in patients with aplastic anemia

To investigate the role of erythropoietin in aplastic anemia, the effects of high titers of recombinant human erythropoietin (rh-Ep) on CFU-E and BFU-E in patients with aplastic anemia were studied in vitro. Colony assays were performed by methylcellulose culture methods added with 1 to 500 units of rh-Ep. In normal bone marrow, the maximum CFU-E colony formation was observed at 2 to 5 units of rh-Ep, and BFU-E at 2 to 10 units. Colonies did not increase by addition of higher titer of rh-Ep to the cultures. In aplastic anemia, the numbers of CFU-E and BFU-E were low at 2 units of rh-Ep in culture system. In most patients with aplastic anemia studied, erythroid colonies were increased in accordance with the increase of rh-Ep added to cultures. These results suggest that the administration of high titers of rh-Ep in vivo may be useful for the improvement of anemia in aplastic anemia.     

Heme metabolism and in vitro erythropoiesis in anemia associated with hypochromic microcytosis

Heme metabolism and in vitro erythropoietic growth (CFU-E, BFU-E) were examined in bone marrow cells taken from two siblings with apparent familial hypochromic microcytic anemia. Bone marrow cells from both patients grew adequate numbers of CFU-E and BFU-E colonies in culture in the presence of erythropoietin. In addition, small numbers of endogenous CFU-E were seen in 7-day cultures. Assays on bone marrow cells taken from both patients revealed that baseline delta-aminolevulinic synthase activity was considerably reduced, but increased six to seven fold (to normal levels) when patients' cells were exposed to pyridoxal phosphate (PLP). In both cases, ferrochelatase and delta-aminolevulinic acid dehydratase activities were normal. Bone marrow heme oxygenase showed no significant differences in activities between normals and patients values in the absence or presence of PLP. In contrast, heme synthesis by patients' bone marrow was less than that of normals. This study demonstrates that bone marrow cells from patients with this rare disorder have some disturbances in heme metabolism, whereas erythropoiesis appeared to be normal when cultured with adequate nutrients in vitro.     

Effect of recombinant hematopoietic growth factors on proliferation of human marrow progenitor cells in serum-deprived liquid culture

We investigated the effects of recombinant interleukin-3 (IL-3), granulocyte-macrophage and granulocyte colony-stimulating factors (GM- CSF and G-CSF), and erythropoietin (Ep) on the number of human hematopoietic progenitors after two to ten days of incubation in liquid cultures deprived of fetal bovine serum (FBS). The source of progenitor cells was normal human marrow depleted of T lymphocytes and/or adherent cells. When adherent cell-depleted marrow was cultured without growth factors, the number of progenitor cells was relatively constant for periods up to eight days. In contrast, a progressive decline in the number of progenitor cells was detected in cultures of nonadherent, T- cell-depleted marrow cells. In both cases, the addition of IL-3 increased by two- to fourfold over input the number of erythroid burst- forming cells (BFU-E) per culture. The number of BFU-E peaked either at day 4 or 8. G-CSF had no effect on the number of progenitor cells per culture. GM-CSF and Ep had no effect in cultures of nonadherent marrow cells but maintained the number of BFU-E in cultures of nonadherent, T- cell-depleted marrow cells. The addition of a neutralizing anti-GM-CSF monoclonal antibody, but not anti-IL-3 neutralizing antiserum, decreased the number of BFU-E in cultures of nonadherent marrow cells. None of the growth factors investigated enhanced the number of GM progenitors to the same degree as the number of BFU-E. However, in cultures of nonadherent, T-cell-depleted marrow cells, IL-3 and GM-CSF maintained the number of GM progenitors up to eight days. These results indicate that IL-3 alone is capable of increasing the number of BFU-E and of maintaining the number of GM progenitors in liquid culture, whereas GM-CSF and Ep are capable of maintaining, but not increasing, BFU-E in this system.     

Mast cell growth factor (c-kit ligand) supports the growth of human multipotential progenitor cells with a high replating potential.

The replating capability of human multipotential (colony-forming unit-granulocyte-erythrocyte-macrophage-megakaryocyte [CFU-GEMM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitors was assessed in vitro as a potential measure of self-renewal using purified, recombinant (r) human (hu) or murine (mu) mast cell growth factor (MGF), a ligand for the c-kit proto-oncogene receptor. Primary cultures of human umbilical cord blood or adult human bone marrow cells were initiated in methylcellulose with erythropoietin (Epo) alone or in combination with rhu interleukin-3 (IL-3) or MGF. Individual day 14 to 18 CFU-GEMM or BFU-E colonies were removed from primary cultures and reseeded into secondary methylcellulose cultures containing a combination of Epo, MGF, and rhu granulocyte-macrophage colony-stimulating factor (GM-CSF). The data showed a high replating efficiency of cord blood and bone marrow CFU-GEMM in response to Epo + MGF in terms of the percentage of colonies that could be replated and the number of secondary colonies formed per replated primary colony. The average number of hematopoietic colonies and clusters apparent from replated cultures of cord blood or bone marrow CFU-GEMM stimulated by Epo + MGF was greater than with Epo + rhuIL-3 or Epo alone. Replated cord blood CFU-GEMM gave rise to CFU-GEMM, BFU-E, and GM colony-forming units (CFU-GM) in secondary cultures. Replated bone marrow CFU-GEMM gave rise mainly to CFU-GM in secondary cultures. A more limited capacity for replating of cord blood and bone marrow BFU-E was observed. These studies show that CFU-GEMM responding to MGF have an enhanced replating potential, which may be promoted by MGF. These studies also support the concept that MGF acts on more primitive progenitors than IL-3.     

In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia. No clear explanation has been given of its defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the nature of this defect we studied the effect of several human recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We observed a defect underlying the early erythroid progenitors, which were unresponsive to several growth factors (erythropoietin, interleukin-3 [IL-3], IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], erythroid potentiating activity), either alone or in association. The production of cytokines was not impaired, and high levels of IL-3 and GM-CSF were found in phytohemagglutinin-leukocyte-conditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic stem cells in DBA patients may be induced to differentiate to the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34+ cell preincubation with IL-3. Addition of the stem cell factor to IL-3 and erythropoietin induces a dramatic in vitro increase in both the number and the size of BFU-E, which also display a normal morphologic terminal differentiation.     

Erythropoiesis in Fanconi's anemia.

Fanconi's anemia (FA) is an autosomal recessive condition in which greater than 90% of the homozygotes develop aplastic anemia. To determine the relation between erythroid progenitors and clinical status, blood and marrow mononuclear cells were cultured in methyl cellulose with erythropoietin, plus other hematopoietic growth factors, and growth in normal oxygen (20%) was compared with growth in low, physiologic oxygen (5%). Peripheral blood cultures were performed from 24 patients, and marrows from six. Patients were classified into six clinical groups. Group 1: Severe aplasia, transfused; one patient; no erythroid progenitors. Group 2: Severe, transfused, androgen unresponsive; one patient; no blood burst-forming units-erythroid (BFU-E). Group 3: Androgen responsive; eight patients, with decreased blood BFU-E. Group 4: Aplastic, about to start treatment; two patients; below normal numbers of colony-forming units-erythroid (CFU-E) and BFU-E. Group 5: Stable, with mild anemia, and/or thrombocytopenia, and/or macrocytosis; seven patients; with below normal numbers of blood BFU-E. Group 6: Hematologically normal; five patients; blood BFU-E low normal to normal. One marrow had normal numbers of CFU-E and BFU-E. Incubation in 5% oxygen doubled CFU-E and BFU-E only in the patients with close to normal or normal growth in 20% oxygen. Hemin and interleukin-3 increased growth slightly in those cultures where there was some growth with erythropoietin alone. Our data show that there is a correlation between current clinical status and in vitro erythropoiesis. Cultures of erythroid progenitors may also be useful predictors of hematologic prognosis in FA, although our follow-up period is too short to prove this hypothesis.     

Reactivation of HbF synthesis in normal adult erythroid bursts by IL-3

Reactivation of HbF synthesis has been reported in normal adult erythroblast colonies ('burst') generated by erythroid progenitors (BFU-E) after seeding peripheral blood mononuclear cells (PBMC) in fetal calf serum-supplemented (FCS+) semisolid cultures stimulated by erythropoietin (Ep). Reactivation is almost totally suppressed when: (i) PMBC are grown in optimized FCS- culture or (ii) PBMC are first stringently depleted of monocytes and then plated in FCS+ medium (i.e. BFU-E growth in FCS+Mo- culture). In either case, addition of biosynthetic granulocyte-macrophage colony stimulating factor (GM-CSF) induces a dose-related increase of relative HbF synthesis up to the level in FCS+ culture. We report that, in FCS- culture of partially purified adult blood BFU-E, treatment with biosynthetic interleukin 3 (IL-3) causes a dose-related rise of relative HbF production in the bursts. A similar phenomenon is observed in FCS+ culture of highly purified BFU-E. The rise of HbF synthesis is seemingly mediated, at least in part, by a direct effect of IL-3 at BFU-E level. It is tentatively concluded that reactivation of HbF in vitro, as well as in a variety of in vivo conditions (i.e. stress erythropoiesis, marrow regeneration), may be at least in part mediated by IL-3 and GM-CSF.     

Erythroid failure in Diamond-Blackfan anemia is characterized by apoptosis

Programmed cell death, also known as apoptosis, is frequently initiated when cells are deprived of specific trophic factors. To investigate if accelerated apoptosis contributes to the pathogenesis of Diamond- Blackfan anemia (DBA), a rare pure red blood cell aplasia of childhood, we studied the effect of erythropoietin (epo) deprivation on erythroid progenitors and precursors from the bone marrow of DBA patients as compared with hematologically normal controls. Apoptosis in response to epo deprivation was evaluated by enumeration of colony-forming unit- erythroid (CFU-E)- and burst-forming unit-erythroid (BFU-E)-derived colonies in plasma clot semisolid culture and by the identification of typical DNA oligosomes by gel electrophoresis from marrow mononuclear cells in liquid culture. In all DBA patients there was a marked decrease in CFU-E- and BFU-E-derived colony formation compared with normal controls at comparable time points of epo deprivation, with a complete loss of CFU-E-derived colonies in semisolid culture by 9 hours of epo deprivation versus 48 hours in controls. The BFU-E-derived colony response to epo deprivation displayed a similar pattern of decrement. Apoptotic changes assessed by the presence of characteristic DNA fragmentation began in the absence of epo deprivation and were readily detected within 3 hours of epo deprivation in DBA cultures versus 9 hours in controls. We conclude that DBA is characterized by accelerated apoptosis as measured by the loss of erythroid progenitor clonogenicity and increased progenitor and precursor DNA fragmentation leading to the formation of characteristic oligosomes, consistent with an intrinsic erythroid-progenitor defect in which increased sensitivity to epo deprivation results in erythroid failure.     

Action of interleukin-3, G-CSF, and GM-CSF on highly enriched human hematopoietic progenitor cells: synergistic interaction of GM-CSF plus G-CSF

Purified preparations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and interleukin 3 (IL-3 or multi-CSF) alone and in combination, have been compared for their stimulatory effects on human granulocyte-macrophage colony forming cells (GM-CFC). In cultures of unseparated normal human bone marrow, the combinations of G-CSF plus IL-3 and GM-CSF plus IL-3 stimulated additive numbers of GM colonies, while GM-CSF plus G-CSF stimulated greater than additive numbers of GM colonies, compared with the sum of the colony formation obtained with each factor alone. Cultures of unseparated bone marrow, harvested from patients four to six days after administration of 5-fluorouracil (5-FU), resulted in additive GM colony formation with GM-CSF plus G-CSF, GM-CSF plus IL-3, and G-CSF plus IL-3. In order to address the possibility of secondary factor involvement in the synergistic interaction of GM-CSF and G-CSF, CD33+/CD34+ colony forming cells were separated from normal and post FU marrow by two color fluorescence activated cell sorting. In cultures of CD33+/CD34+ cells the combination of GM-CSF plus G-CSF stimulated a synergistic increase in GM colonies while GM-CSF plus IL-3 stimulated additive numbers of colonies. These results suggest that GM-CSF, G-CSF, and IL-3 stimulate distinct populations of GM-CFC. Furthermore GM-CSF and G-CSF interact synergistically and this action is a direct effect on progenitor cells not stimulated by GM-CSF or G-CSF alone.     

Interleukin-10 inhibits burst-forming unit-erythroid growth by suppression of endogenous granulocyte-macrophage colony-stimulating factor production from T cells

Numerous cytokines released from accessory cells have been shown to exert either stimulatory or inhibitory growth signals on burst-forming unit-erythroid (BFU-E) growth. Because of its cytokine synthesis-inhibiting effects on T cells and monocytes, interleukin-10 (IL-10) may be a potential candidate for indirectly affecting erythropoiesis. We investigated the effects of IL-10 on BFU-E growth from normal human peripheral blood mononuclear cells (PBMC) using a clonogenic progenitor cell assay. The addition of recombinant human IL-10 to cultures containing recombinant human erythropoietin suppressed BFU-E growth in a dose-dependent manner (by 55.2%, range 47.3-63.3%, p < 0.01, at 10 ng/mL). In contrast, no inhibitory effect of IL-10 was seen when cultivating highly enriched CD34+ cells. BFU-E growth from PBMC also was markedly suppressed in the presence of a neutralizing anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (by 48.7%, range 32.9-61.2% inhibition,p < 0.01), but not by neutralizing antibodies against granulocyte colony-stimulating factor and interleukin-3. This suggests a stimulatory role of endogenously released GM-CSF on BFU-E formation. Also, the addition of exogenous GM-CSF completely restored IL-10-induced suppression of BFU-E growth. To determine the cellular source of GM-CSF production, we analyzed GM-CSF levels in suspension cultures containing PBMC that were either depleted of monocytes or T cells. Monocyte-depleted PBMC showed spontaneous production of increasing amounts of GM-CSF on days 3, 5, and 7, respectively, which could be suppressed by IL-10, whereas GM-CSF levels did not increase in cultures containing T-cell-depleted PBMC. Our data indicate that IL-10 inhibits the growth of erythroid progenitor cells in vitro, most likely by suppression of endogenous GM-CSF production from T cells.     

Anaemia of chronic disease in rheumatoid arthritis: effect of the blunted response to erythropoietin and of interleukin 1 production by marrow macrophages.

Anaemia in rheumatoid arthritis (RA) is a common and debilitating complication. The most common causes of this anaemia are iron deficiency and anaemia of chronic disease. Investigations have suggested that interleukin 1 (IL-1) or tumour necrosis factor (TNF), or both, from monocytes associated with chronic inflammation are responsible for the anaemia of chronic disease. On bone marrow examination anaemia of chronic disease is characterised by the diversion of iron from the erythropoietic compartment into marrow macrophages. This phenomenon is termed failure of iron utilisation. In this study, CFU-E (colony forming unit erythroid; late red cell precursors) and BFU-E (burst forming unit erythroid; early red cell precursors) stem cells were cultured from 10 normal marrow samples and 12 marrow samples from patients with RA with iron deficiency anaemia and 10 samples from patients with RA with failure of iron utilisation. All patients with RA were anaemic (haemoglobin less than 100 g/l), Potential accessory or inhibitory cells of erythropoiesis (CD4, CD8, or CD14 positive cells) were removed before culture. Control marrow samples were studied in a similar manner. Normal marrow samples yielded 377 (17) CFU-E and 133 (6) BFU-E (mean (SD)) colonies for each 2 x 10(5) light density cells plated. CD4 ablation caused reductions of 62 and 100% in CFU-E and BFU-E colonies respectively. CD14 removal resulted in considerable but lesser reductions of 46% for CFU-E and 25% for BFU-E. In both groups of patients with RA, CFU-E colony numbers were significantly lower than those seen in normal control subjects, 293 (17) for patients with iron deficiency anaemia and 242 (35) for patients with failure of iron utilisation. BFU-E colony numbers were 102 (13) and 108 (20) respectively. In patients with RA, CD4 removal caused a significantly greater loss of CFU-E colonies compared with normal control subjects. Cytolysis of CD14 positive cells caused a reduction in CFU-E colonies in the two RA groups which was similar to that seen in normal subjects. In conclusion, patients with RA seem to have fewer CFU-E progenitors but essentially normal numbers of BFU-E stem cells. Our data suggest a stimulatory role for marrow CD4 and CD14 cells in erythropoiesis in patients with RA. Monocytes-macrophages (CD14 positive) are known to be producers of IL-1 or TNF, or both, however, the predicted increase in the CFU-E colonies on removal of CD14 cells is not seen. Therefore, if IL-1 or TNF, or both, are responsible for the impairment of erythropoiesis in patients with RA, marrow macrophages are unlikely to be the source. Moreover, these results indicate the probability of erythropoietin resistance on the basis of diminished CFU-E colony formation in patients with RA.     

Transient erythroblastopenia of childhood: Varied pathogenesis

The mechanism of anemia in four patients with transient red cell aplasia of childhood (“erythroblastopenia”) was studied at the time of diagnosis by assessing the colony growth of marrow erythroid progenitors in methylcellulose tissue cultures. Marrow from Patient 1 yielded high normal numbers of BFU-E colonies that were completely abolished on addition of autologous serum or IgG. Patient 2 had normal BFU-E growth that markedly declined when autologous serum or IgM was added to the cultures, but growth remained unchanged with added autologous IgG or peripheral blood mononuclear cells (PBMC). Marrow from Patient 3 yielded low CFU-E and BFU-E numbers with standard plating techniques, but colonies strikingly increased when marrow fractions from an albumin density gradient were cultured. PBMC from Patient 3 suppressed control marrow CFU-E and BFU-E, but serum had no effect. Patient 4 had normal CFU-E and BFU-E that increased with autologous serum and remained unchanged with autologous PBMC. We conclude that the red cell aplasia in Patients 1, 2, and 3 was due to suppressed erythropoiesis via IgG, IgM, and cell-mediated inhibition, respectively. In contrast, in Patient 4 no immune mechanism was demonstrated. Whereas transient red cell aplasia has a uniform clinical presentation, there are at least four pathogenetic mechanisms that can be detected in vitro.     

Erythropoietin alone induces erythroid burst formation by human embryonic but not adult BFU-E in unicellular serum-free culture [see comments]

Erythroid progenitors (BFU-E) from adult human peripheral blood generate erythroid bursts in semisolid culture supplemented with at least two growth factors, ie, erythropoietin (Ep) and interleukin-3 (IL- 3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). We have analyzed the hematopoietin(s) requirement of human embryonic BFU- E, as compared to that of adult peripheral blood progenitors: This was basically evaluated in fetal calf serum-free (FCS-) methylcellulose culture of partially or highly purified progenitors treated with human recombinant hemopoietins. At a low seeding concentration (2 x 10(3) cells/dish) purified embryonic BFU-E generated erythroid bursts when treated only with Ep: Further addition of IL-3 or GM-CSF had no effect on BFU-E cloning efficiency, although the size of bursts was increased in a dose-dependent manner, particularly with IL-3. At a similar seeding concentration (ie, 10(3) cells/dish), purified adult BFU-E efficiently generated erythroid bursts in the presence of Ep and GM-CSF or IL-3, while only few small erythroid colonies were observed in the presence of Ep alone. In a final series of experiments, unicellular FCS- cultures of purified embryonic BFU-E gave rise to erythroid bursts in the presence of Ep alone. Furthermore, the cloning efficiency induced by Ep was unmodified by further addition of GM-CSF or IL-3. Unicellular FCS- cultures of highly purified adult peripheral blood progenitors generated no erythroid bursts in the presence of Ep alone. The addition of GM-CSF or IL-3 was required to generate BFU-E colonies. These studies indicate that in human embryonic life, BFU-E require only Ep for efficient erythroid burst formation, while IL-3 and GM-CSF essentially enhance the proliferation of early erythropoietic precursors.     

Combined and sequential effects of human IL-3 and GM-CSF on the proliferation of CD34+ hematopoietic cells from cord blood

The proliferative effects of recombinant human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were investigated in semi-solid and liquid cultures of purified CD34+ hematopoietic cells obtained from umbilical cord blood. No important differences in overall cloning efficiencies in response to IL-3 or GM- CSF were observed in semi-solid medium in the presence of erythropoietin (Ep). However, GM-CSF was less effective for the development of erythroid bursts (BFU-E), and only IL-3 was observed to induce significant numbers of mixed-erythroid colonies (E-MIX). Both IL- 3 and GM-CSF also induced proliferation of CD34+ in liquid cultures. Proliferative responses to IL-3 were found to be more rapid and stronger than to GM-CSF, although the number of initial responsive cells as judged by autoradiography were comparable. Enhanced proliferation of CD34+ cells both in semi-solid and liquid cultures was obtained in the presence of combinations of IL-3 and GM-CSF. The responses observed were less than additive, with the exception of the development of eosinophil colonies and clusters, where IL-3 and GM-CSF were found to act synergistically. In secondary cultures, proliferative responses to GM-CSF were strongly enhanced by preculture of CD34+ cells in IL-3 for four to 11 days, and to a lesser extent by preculture in GM- CSF. Finally, responses to IL-3 were not affected by preculture of CD34+ cells in the presence of GM-CSF. Our results indicate that there is a wide overlap of cells capable of proliferating either in response to IL-3 or to GM-CSF within the cord blood CD34+ compartment. However, differences in primary proliferation kinetics and increased responsiveness to GM-CSF following preculture suggest the importance of a sequential action of IL-3 and GM-CSF in the expansion of CD34+ cells.     

Studies on hematopoietic stem cells: XI. Lack of erythroid burst-forming units (BFU-E) in patients with aplastic anemia.

The marrow concentration of erythropoietic precursors was examined in normal donors and patients with idiopathic aplastic anemia using a plasma clot culture system. On time course observations the heterogeneity of human erythroid precursors assayable in culture was demonstrated. To evaluate human erythropoiesis in vitro, the benzidine-positive colonies were divided into three groups: small colony, containing 8-50 cells; medium-sized colony, containing 50-500 cells; and large colony, containing more than 500 cells. The majority of the large colonies assumed the morphology of erythropietic bursts (BFU-E) consisted of several subcolonies. The small colonies were counted as CFU-E1, the medium-sized as CFU-E2, and the large as BFU-E to evaluate the erythroid precursor cell compartment in aplastic anemia. The marrow concentration of CFU-E1 and CFU-E2 was shown to be quantitatively diminished in aplastic anemia. In addition, there was no ability of the marrow cells from aplastic patieints to grow BFU-E in vitro even in the presence of a large dose of erythropoietin. This lack of BFU-E colony growth may play an important role in the mechanism of the erythropoietic deficiency in aplastic anemia.     

In vitro growth of human fetal CD34+ cells in the presence of various combinations of recombinant cytokines under serum-free culture conditions

Summary. CD34⁺ cells were purified from midtrimester human fetal blood and adult bone marrow samples and seeded in serum-free fibrin-clot cultures in order to evaluate the number and the responsiveness to recombinant cytokines of pluripotent (CFU-GEMM), erythroid (BFU-E), megakaryocyte (BFU-meg and CFU-meg) and granulocyte/macrophage (CFU-GM) haemopoietic progenitor cells.
The number of the different haemopoietic progenitors/1 × 10³ CD34⁺ cells, except CFU-meg, was significantly higher in fetal blood than in adult bone marrow in cultures stimulated by any combination of cytokines including interleukin-3 (IL-3), granulocyte/macrophage colony stimulating factor (GM-CSF) or stem cell factor (SCF) plus erythropoietin (Epo). Nevertheless, whereas adult BFU-E showed a maximal growth in the presence of Epo plus IL-3 or Epo plus SCF, fetal BFU-E showed an optimal growth in the presence of Epo alone, the sensitivity of fetal BFU-E to suboptimal concentrations of Epo being approximately 10–15-fold higher than that of adult BFU-E. Addition of optimal concentrations of IL-3, GM-CSF or SCF, alone or in various combinations, to Epocontaining cultures induced a significant increase in both the number and size of fetal CFU-GEMM, and CFU-GM, and a parallel decrease of fetal BFU-E. Finally, SCF potently syner-gized with IL-3 in increasing the growth of both classes of fetal megakaryocyte progenitors, BFU-meg and CFU-meg.     

Polycythemia vera. II. Hypersensitivity of bone marrow erythroid, granulocyte-macrophage, and megakaryocyte progenitor cells to interleukin-3 and granulocyte-macrophage colony-stimulating factor.

Polycythemia vera (PV) is a clonal disease of the hematopoietic stem cell characterized by a hyperplasia of marrow erythropoiesis, granulocytopoiesis, and megakaryocytopoiesis. We previously reported that highly purified PV blood burst-forming units-erythroid (BFU-E) are hypersensitive to recombinant human interleukin-3 (rIL-3). Because these cells may be only a subset, and not representative of marrow progenitors, we have now studied partially purified marrow hematopoietic progenitor cells. Dose-response experiments with PV marrow BFU-E showed a 38-fold increase in sensitivity to rIL-3 and a 4.3-fold increase in sensitivity to recombinant human erythropoietin (rEpo) compared with normal marrow BFU-E. In addition, PV marrow colony-forming units-granulocyte-macrophage (CFU-GM) and CFU-megakaryocyte (CFU-MK) also showed a marked hypersensitivity to rIL-3 and to human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Dose-response curves with rGM-CSF and blood BFU-E showed a 48-fold increase in sensitivity. No effect of rIL-4, rIL-6, human recombinant granulocyte-CSF (rG-CSF), or macrophage-CSF (rM-CSF) was evident, nor was there any effect of PV cell-conditioned medium on normal BFU-E, when compared with normal cell-conditioned medium. Autoradiography with 125I-rEpo showed an increase in Epo receptors after maturation of PV BFU-E to CFU-E similar to that shown with normal BFU-E, but no increase of specific binding of 125I-rIL-3 by PV CD34+ cells was seen compared with normal CD34+ cells. These studies show that PV marrow hematopoietic progenitor cells are hypersensitive to rIL-3 and rGM-CSF, similar to PV blood BFU-E. While the mechanism does not appear to be due to enhanced binding of rIL-3, the hypersensitivity of PV progenitor cells to IL-3 and GM-CSF may be a key factor in the pathogenesis of PV.