Granulocyte-macrophage colony-stimulating factor reactivates fetal hemoglobin synthesis in erythroblast clones from normal adults

Reactivation of fetal hemoglobin (HbF, alpha 2 gamma 2) synthesis was previously reported in normal human adult erythroblast colonies ("bursts") generated by erythroid progenitors (BFU-E) in fetal calf serum-supplemented (FCS+) semisolid cultures stimulated with erythropoietin (Ep). Our studies focused on the reactivation of HbF synthesis in normal adult erythroid bursts generated by peripheral blood mononuclear cells (PBMCs) seeded in FCS+ methylcellulose culture. Reactivation is almost totally suppressed when (a) PBMCs are grown in optimized FCS- culture, or (b) PBMCs are first stringently depleted of monocytes and then plated in FCS+ medium (ie, BFU-E growth in FCS+ Mo- culture). In both experimental conditions, the proliferation of lymphocytes and macrophages interspersed among colonies is drastically reduced, and the cloning efficiency of granulocyte-macrophage (GM) progenitors is sharply diminished. In either case, addition of biosynthetic GM colony-stimulating factor (GM-CSF) induces a dose-related increase of HbF synthesis up to the level in FCS+ culture, with even more elevated values on delayed addition of Ep. A dose-related increase was also observed in erythroblast clones generated by highly purified BFU-E. These results suggest that reactivation of HbF synthesis in normal adults is at least in part mediated by GM-CSF. Furthermore, they imply intriguing hypotheses on the mechanism(s) of perinatal Hb switching. Finally, they raise the possibility of reactivation of HbF synthesis in beta-thalassemia and sickle cell anemia by GM-CSF therapy.     

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.     

c-kit ligand reactivates fetal hemoglobin synthesis in serum-free culture of stringently purified normal adult burst-forming unit- erythroid

We have analyzed the reactivation of fetal hemoglobin (HbF) synthesis under rigorous in vitro conditions, ie, in mature erythroblasts generated by erythroid burst-forming units (BFU-E) stringently purified from normal adult peripheral blood and grown in fetal calf serum(FCS)- free semisolid or liquid phase culture. In clonogenetic dishes, graded amounts of c-kit ligand (KL) were added together with saturating levels of erythropoietin (Ep) and variable amounts of interleukin-3 and granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF), ie, high or low level, or no IL-3/GM-CSF addition. In all conditions, KL induced a sharp, dose-dependent increase in the percentage of F cells and HbF content from nearly normal levels (< 10% and < 2.5%, respectively, at 0.1 and 1 ng/mL) up to 40% to 50% and 10% to 15% at 100 to 200 ng/mL. This increase was not associated with significant differences of burst number or stage of maturation at the time of analysis (as evaluated on the basis of percent mature erythroblasts and Hb content per cell). However, the KL-induced reactivation of HbF synthesis was strictly and directly correlated with a sharp increase of colony size, ie, cell number per burst. Addition of large amounts of IL- 3 and GM-CSF (10 to 100 U and 1 to 10 ng/mL, respectively) significantly potentiated the KL-induced reactivation of HbF, as compared with low levels (0.1 U and 0.01 to 0.1 ng) or no addition of these growth factors: this increase was highly significant at low KL doses (ie, 1 to 10 ng/mL). Single-burst analysis showed that the KL- induced HbF reactivation occurs homogeneously in the erythroid colonies within each of these culture conditions. We have analyzed the effect of KL in liquid phase BFU-E culture treated with the IL-3/GM-CSF/Ep combination at sequential times until terminal erythroid maturation: KL causes a sharp increase in the percentage of F cells and HbF content in all stages of maturation, whereas the IL-3/GM-CSF/Ep combination alone has a markedly lower effect. These results suggest that KL plays a key role in the reactivation of HbF synthesis in adult life, whereas IL- 3/GM-CSF potentiate this effect at low KL levels. The KL-induced HbF reactivation is seemingly related to an enhanced proliferation of erythroid progenitors in the erythropoietic differentiation pathway.     

Inhomogeneity of the circulating BFU-E regulation in sickle cell anaemia: accessory cells properties and BFU-E growth factor response pattern

Summary. Sickle cell anaemia (SS) patients with low (<9%) HbF levels (LFSS) are characterized by an increased number of circulating BFU-E in active DNA synthesis, and release of burst promoting activity (BPA) by unstimulated low density (LD) adherent cells. In contrast, circulating BFU-E from SS patients with high (>9%) HbF levels (HFSS) are normal in number, largely in resting phase, and their LD cells do not release BPA-like activity.
We report now that in LFSS patients, adherent cell depletion decreases BFU-E growth in culture and apparent BFU-E cycling. Furthermore, addition of conditioned media (CM) from LD cells of LFSS patients restored cycling BFU-E expression in culture. Neutralization analysis with anti-GM-CSF antibody demonstrated that GM-CSF is, at least, one factor responsible for BPA activity present in this CM. Thus, GM-CSF is constitutively produced by unstimulated monocytes in LFSS patients. In contrast, HFSS patients' adherent cell depletion increases cycling of BFU-E in culture. CM from HFSS patients inhibits BFU-E expression in culture. Hence, LD adherent cells from HFSS patients may release a yet unknown inhibitor factor(s). In addition, we report a distinct response pattern in SS patients' BFU-E to growth factor (GM-CSF, IL-3): (a) LFSS patients have a BFU-E population, equally responsive to GM-CSF and IL-3; (b) HFSS patients, have a subset of BFU-E exclusively dependent on IL-3 (20–40% of the circulating BFU-E). This pattern is very similar to that of normal BFU-E.
In conclusion, BFU-E from LFSS patients represent an actively proliferating population, equally responsive to GM-CSF and IL-3, controlled by constitutively produced GM-CSF, suggesting a unique BFU-E behaviour in SS patients with low HbF levels and high haemopoietic stress. The heterogeneous regulation of BFU-E in SS disease seems to be the epiphenomenon of HbF levels, and not vice versa.     

Erythroid burst-promoting activity of purified recombinant human GM-CSF and interleukin-3: studies with anti-GM-CSF and anti-IL-3 sera and studies in serum-free cultures

We studied the erythroid burst-promoting activity (BPA) of recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) and Interleukin-3 (IL-3) with two experimental approaches. First we studied the effects of polyclonal antisera prepared against human GM-CSF and gibbon IL-3 on colony formation from 1,000 bone marrow null cells/dish in serum-containing culture. Both GM-CSF and IL-3 independently enhanced erythroid burst formation; however, IL-3 showed more BPA activity than GM-CSF. These data are in agreement with an emerging view that the primary targets of IL-3 are primitive progenitors and that the targets of GM-CSF are intermediate progenitors, including erythroid burst-forming units (BFU-E). The proliferation of one population of BFU- E was independent of GM-CSF or IL-3. To characterize this population of BFU-E further, we developed a serum-free culture assay for the purified progenitors by incorporating insulin-like growth factor-1 (IGF-1) to the serum-free medium. The development of erythroid bursts was supported by IL-3, IGF-1, and erythropoietin (Ep) in a serum-free culture system and to a lesser extent by the combination of GM-CSF, IGF- 1, and Ep. Although the burst-promoting ability of GM-CSF and IL-3 was again demonstrated in this system, unlike serum-containing culture Ep alone did not support burst formation. These results indicate that when fetal calf serum (FCS) is present, the culture system contains BPA that is not GM-CSF or IL-3.     

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.     

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.     

Globin synthesis in erythroid bursts that mature sequentially in culture. I. Studies in cultures of adult peripheral blood BFU-Es

To study whether the culture time at which the burst populations mature influences the expression of fetal hemoglobin in bursts, we measured hemoglobin synthesis in cohorts of fully hemoglobinized erythroid bursts maturing sequentially in cultures of adult peripheral blood BFU- Es. In 13 of 15 experiments, a decline in gamma/gamma + beta ratio was noted as the culture time advanced. On the average, erythroid bursts that mature during the third culture week showed lower levels of fetal Hb synthesis compared to bursts that are already mature in the second culture week. The decline of gamma/gamma + beta ratio with culture time was also noted in erythroid bursts composed of immature erythroblasts. The enhanced HbF formation in peripheral blood BFU-E cultures is thus most pronounced among the bursts that become hemoglobinized early, and there is a tendency for normalization of HbF synthesis in bursts that mature in late culture days. These results can be interpreted by several alternatives, including the possibility that the expression of high HbF levels in the early days of adult BFU-E cultures is a reflection of premature commitment to terminal differentiation of progenitors that possess an active HbF program. The present data indicate that the variation of HbF synthesis with culture time should be taken into consideration when the influence of various culture conditions of HbF synthesis is studied in BFU-E cultures.     

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.     

Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures.

Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady-state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units-erythroid (BFU-E) colonies. There was a high correlation of gamma-globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma-globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma-globin expression in adult erythroid progenitor cell cultures.     

Evidence for direct action of human biosynthetic (recombinant) GM-CSF on erythroid progenitors in serum-free culture

The biologic activity of human biosynthetic granulocyte-monocyte colony stimulating factor (GM-CSF) was investigated in serum-free culture of erythroid progenitors derived from adult peripheral blood. The morphology of erythroid bursts and the cloning efficiency of BFU-E under serum-free conditions were similar to those observed in dishes with fetal bovine serum (FBS). For these experiments, progenitor cells were partially purified by Ficoll-Paque density centrifugation, adherence to a plastic surface, and complement-mediated cytotoxicity of Leu-1+ elements. For some studies, blastlike cells were harvested directly from 6-day-old semisolid cultures. In serum-free culture of the light-density cell fraction, biosynthetic erythropoietin (Ep) was sufficient for formation of pure and mixed erythroid colonies whereas GM-CSF was required for granulocyte-monocytic colonies. When adherent and Leu-1+ cells were removed, or when in vitro differentiated blast cells were used as a source of progenitors, neither Ep or GM-CSF alone induced colony formation. In dishes supplemented with both growth factors, erythroid bursts were detected. Although the presence of GM- CSF alone did not induce formation of any colony or clusters, BFU-E were recorded when Ep was added 8 days later, suggesting that BFU-E could be maintained. Terminal maturation of the resulting erythroid bursts was delayed by 8 days. These results provide evidence that GM- CSF acts directly on early erythroid progenitors. Furthermore, they suggest that both Ep and GM-CSF are necessary to start the differentiation process.     

Augmentation of fetal hemoglobin (HbF) synthesis in culture by human erythropoietic precursors in the marrow and peripheral blood: studies in sickle cell anemia and nonhemoglobinopathic adults

We cultured marrow and peripheral blood erythropoietic precrusors in methylcellulose clonal assay and measured the synthetic rates of HbA, A2, F, and S in patients with and without sickle cell anemia. Hb was labeled with 14C-amino acid in culture, separated by slab gel isoelectric focusing techniques, and quantitated by autoradiographic methods. Comparison of marrow late (CFU-E) and early (BFU-E) precursors from patients without hemoglobinopathies showed that preferential synthesis of HbF is limited to early precursors. Simultaneous examinations of Hb synthesis by blood and marrow early erythropoietic precursors confirmed the similarity of the biosynthetic capabilities of the precursors from the two sources. Increasing concentrations of erythropoietin (Ep) in culture corresponded with increases in the percentages of HbF synthesized by blood BFU-E of normal individuals. HbF biosynthesis by blood BFU-E from sickle cell anemia patients was significantly higher than that synthesized by nonanemic individuals and showed significant individual variations. HbF synthesis in patients with sickle cell anemia was partially dependent on Ep concentrations in culture. Cell culture of circulating erythropoietic precursors in man appears to provide a unique tool for studying the control mechanisms of Hb synthesis in man.     

The role of macrophages in the regulation of erythroid colony growth in vitro.

Depletion of macrophages from murine marrow by the use of a monoclonal anti-macrophage antibody resulted in a significant increase in the number of erythroid burst forming units (BFU-E). This increase could be neutralized by the addition back to culture of macrophages or macrophage conditioned medium indicating that the suppression was mediated by soluble factors. To further characterize this effect, the addition to culture, either alone or in combination, of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the growth of BFU-E and the colony-forming unit granulocyte-macrophage (CFU-GM) was examined in macrophage-containing and macrophage-depleted cultures. The addition of IL-1 alpha to culture stimulated the release of both TNF alpha and GM-CSF and acted synergistically with both cytokines, resulting in a dose-dependent suppression of BFU-E and stimulation of CFU-GM growth. The increase in CFU-GM caused by the addition of IL-1 alpha was mediated by GM-CSF but not by TNF alpha as the increase was prevented by the addition of a monoclonal anti-GM-CSF antibody but not by anti-TNF alpha. When either TNF alpha or GM-CSF was neutralized by monoclonal antibodies the addition of IL-1 alpha resulted in a significant increase in BFU-E growth. The addition of GM-CSF to culture caused a dose-dependent suppression of BFU-E that was mediated by TNF alpha, as colony number was not reduced when GM-CSF and a monoclonal anti-TNF alpha antibody were simultaneously added to culture. TNF alpha-induced suppression of BFU-E only occurred in the presence of macrophages. In macrophage-depleted cultures, a dose-dependent suppression of BFU-E could be induced if subinhibitory concentrations of IL-1 alpha or GM-CSF were simultaneously added with increasing concentrations of TNF alpha. The effects of IL-1 alpha or GM-CSF and TNF alpha were markedly synergistic so that the doses required to induce suppression when added simultaneously was only 10% of that required when either were added to culture alone. Suppression of BFU-E by GM-CSF or the combined addition of GM-CSF and TNF alpha did not require IL-1 alpha because inhibition was not neutralized by the addition of anti-IL-1 alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of dexamethasone on fetal hemoglobin synthesis in peripheral blood erythroid burst-forming units

Colonies derived from erythroid burst-forming units (BFU-E) synthesize fetal hemoglobin (HbF) in amounts that far exceed in vivo levels. There is some evidence that HbF synthesis is controlled at the level of a primitive erythroid precursor cell. Dexamethasone may potentiate the development of BFU-E. Since a means of augmenting HbF production in sickle cell anemia or severe β-thalassemia would be of great therapeutic value, we studied the effects of dexamethasone on HbF and γ-globin chain synthesis in BFU-E from patients with sickle cell anemia and controls. HbF was measured by radioimmunoassay of BFU-E lysate and γ-chain synthesis by the incorporation of ³H-leucine into globin, which was then purified by gel filtration and column chromatography. Dexamethasone (10^?9 M) produced an increase in the number of BFU-E in 16 of 19 subjects when compared with numbers of BFU-E cultured with only erythropoietin. The individual BFU-E were larger and contained more subcolonies. Dexamethasone did not increase HbF or γ-chain synthesis, and there was no relationship between increased proliferation of BFU-E and augmented HbF production. Thus, although dexamethasone augmented the development of erythroid bursts, there was no increment in HbF.     

Effect of interleukin-9 on clonogenic maturation and cell-cycle status of fetal and adult hematopoietic progenitors

We assessed the effect of interleukin-9 (IL-9) on clonogenic maturation and cell-cycle status of hematopoietic progenitors of fetal (umbilical cord blood) and adult (bone marrow) origin. As a single agent IL-9 supported, in a concentration-dependent fashion, maturation of burst-forming units-erythroid (BFU-E) of adult and fetal origin. However, only 1/3 the number of adult BFU-E colonies developed, as did in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and only 1/6 the number developed as did in response to IL-3. In contrast, the effect of IL-9 on fetal BFU-E colonies was equal to that of GM-CSF and IL-3. Synergistic effects of IL-9 with low concentrations (0.1 ng/mL) of GM-CSF and IL-3 were seen on adult BFU-E colony formation, but no effect was apparent at higher concentrations (1.0 ng/mL). In contrast, using fetal cells, synergistic effects of IL-9 with low and high concentrations of GM-CSF and IL-3 were apparent. Addition of IL-9 to plates containing fetal cells plus GM-CSF and IL-3 not only resulted in more BFU-E colonies, but also in more multicentered (greater than or equal to 10 individual centers) colonies, and more cells per colony. IL-9 had a wider spectrum of action on progenitors of fetal origin than on progenitors of adult origin, supporting the generation of fetal multipotent colony-forming unit (CFU)-Mix and CFU-GM colonies. Incubation with IL-9 did not accelerate cycling of adult or fetal BFU-E, CFU-Mix, or CFU-GM to the extent observed after incubation with IL-6. Thus, IL-9 primarily supported maturation of erythroid progenitors of adult origin, and its addition to plates containing GM-CSF and IL-3 (1.0 ng/mL) did not result in maturation of additional clones. In contrast, IL-9 had a wider spectrum of action on fetal progenitors and, when combined with IL-3 and GM-CSF, resulted in clonogenic maturation of progenitors that did not undergo maturation after stimulation with IL-3 and GM-CSF.     

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.     

Dual action of retinoic acid on human embryonic/fetal hematopoiesis: blockade of primitive progenitor proliferation and shift from multipotent/erythroid/monocytic to granulocytic differentiation program

In preliminary studies, we have analyzed the hematopoietic growth factor (HGF) requirement of hematopoietic progenitor cells (HPCs) purified from embryonic-fetal liver (FL) and grown in fetal calf serum- supplemented (FCS+) clonogenic culture. The key role of erythropoietin (Epo) for colony formation by early erythroid progenitors (burst- forming units-erythroid [BFU-E]) has been confirmed. Furthermore, in the absence of exogenous HGFs, FL monocytic progenitors (colony-forming unit monocyte [CFU-M]) generate large colonies exclusively composed of monocytes-macrophages; these colonies are absent in FCS- clonogenic culture. On this basis, we have investigated the role of all-trans retinoic acid (ATRA) and its isomer 9-cis RA in FL hematopoiesis. Both compounds modulate the growth of purified FL HPCs, which show a dose- dependent shift from mixed/erythroid/ monocytic to granulocytic colony formation. Studies on unicellular and paired daughter cell culture unequivocally indicate that the shift is mediated by modulation of the HPC differentiation program to the granulopoietic pathway (rather than RA-induced down-modulation of multipotent/ erythroid/monocytic HPC growth coupled with recruitment of granulocytic HPCs). ATRA and 9-cis RA also exert their effect on the proliferation of primitive HPCs (high- proliferative potential colony-forming cells [HPP-CFCs]) and putative hematopoietic stem cells (HSCs; assayed in Dexter-type long-term culture). High concentrations of either compound (1) drastically reduced the number of primary HPP-CFC colonies and totally abolished their recloning capacity and (2) inhibited HSC proliferation. It is crucial that these results mirror recent observations indicating that murine adult HPCs transduced with dominant negative ATRA receptor (RAR) gene are immortalized and show a selective blockade of granulocytic differentiation. Altogether, these results suggest that ATRA/9-cis RA may play a key role in FL hematopoiesis via a dual effect hypothetically mediated by interaction with the RAR/RXR heterodimer, ie, inhibition of HSC/ primitive HPC proliferation and induction of CFU- GEMM/ BFU-E/CFU-M shift from the multipotent/erythroid/monocytic to the granulocytic-neutrophilic differentiation program.     

Human interleukin-9 supports formation of a subpopulation of erythroid bursts that are responsive to interleukin-3.

We have investigated the biological activities of recombinant human interleukin-9 (IL-9) on enriched hematopoietic progenitors, alone or in combination with other cytokines, including Epo, G-CSF, IL-3, and GM-CSF, under serum-containing and serum-free cultures. IL-9 alone did not support colony formation. However, IL-9 plus Epo induced erythroid burst (BFU-E) formation derived from peripheral blood (PB) progenitors. Delayed addition experiments demonstrated that a part of bone marrow (BM) derived BFU-E, which seems to be immature, only responded to IL-9 and formed erythroid bursts. The burst-promoting activity (BPA) of IL-9 was confirmed using neutralizing aIL-3, aGM-CSF, and aIL-9 antisera and serum-free culture. IL-9 supported a part of BFU-E population that respond to IL-3, which was almost identical to the number of BFU-E supported by GM-CSF. IL-9 had no additive effect on erythroid and mixed colony formation supported by IL-3. In contrast, IL-9 showed an additive effect on erythroid burst formation supported by GM-CSF in serum-free culture. These data suggest that IL-9 and GM-CSF act on distinct IL-3-responsive BFU-E population. In addition, delayed addition experiment clearly demonstrated that IL-9 supports survival and the early stage of proliferation of BFU-E. These results led us to propose that IL-9 possibly acts as a BPA and selectively supports a subpopulation of early class of BFU-E that respond to IL-3.     

Expansion of granulocyte colony-stimulating factor/chemotherapy-mobilized CD34+ hematopoietic progenitors: role of granulocyte-macrophage colony-stimulating factor/erythropoietin hybrid protein (MEN11303) and interleukin-15

Ex vivo stroma-free static liquid cultures of granulocyte colony-stimulating factor (G-CSF)/chemotherapy-mobilized CD34+ cells were established from patients with epithelial solid tumors. Different culture conditions were generated by adding G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt3 ligand (Flt3), megakaryocyte growth and development factor (Peg-rHuMGDF), GM-CSF/erythropoietin (EPO) hybrid protein (MEN11303), and interleukin-15 (IL-15) to the basic stem cell factor (SCF) + interleukin-3 (IL-3) + EPO combination. This study showed that, among the nine different combinations tested in our 5% autologous plasma-containing cultures, only those containing IL-3/SCF/Flt3/MEN11303 and IL-3/SCF/Flt3/MEN11303/IL-15 significantly expanded colony-forming unit granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), long-term culture-initiating cells (LTC-IC), CD34+, and CD34+/CD38- cells after 14 days of culture. Particularly, the addition of IL-15 to IL-3/SCF/Flt3/MEN11303 combination produced a significant increase of LTC-IC, with an average 26-fold amplification as compared to input cells, without any detrimental effect on CFU-GM and BFU-E expansion. This combination also produced a statistically significant 3.6-fold expansion of primitive CD34+/CD38- cells. Moreover, this study confirms the previously described erythropoietic effect of MEN11303, which, in our experience, was the only factor capable of expanding BFU-E. Compared to equimolar concentrations of GM-CSF and EPO, MEN11303 hybrid protein showed a significantly higher capacity of expanding CFU-GM, BFU-E, LTC-IC, CD34+, and CD34+/CD38- cells when these cytokines were tested in combination with IL-3/SCF/Flt3. These cultures indicated that Peg-rHuMGDF addition to IL-3/SCF/EPO/Flt3 does not affect CFU-GM and BFU-E expansion but, unlike G-CSF or GM-CSF, it does not decrease the ability of Flt3 to expand primitive LTC-IC. These studies indicate that, starting from G-CSF/chemotherapy-mobilized CD34+ cells, concomitant expansion of primitive LTC-IC, CFU-GM, BFU-E, CD34+, and CD34+/CD38- cells is feasible in simple stroma-free static liquid cultures, provided IL-3/SCF/Flt3/MEN11303/IL-15 combination is used as expanding cocktail in the presence of 5% autologous plasma.     

Serum-free culture of enriched hematopoietic progenitors reflects physiologic levels of fetal hemoglobin biosynthesis.

Adult erythroid progenitors produce significantly higher fetal hemoglobin (HbF) levels in cultures containing fetal calf serum (FCS) and erythropoietin (Ep) than in vivo. The precise mechanisms for this increased HbF production in culture have not been elucidated. We examined HbF biosynthesis by enriched human progenitors in serum-free (SF) culture. We measured globin chain biosynthesis by combination of isoelectric focusing and autoradiography and examined percent nucleated erythrocytes containing HbF (%FNRBC) using microscopic immunodiffusion. CD34 (My10)-positive marrow cells from a normal subject yielded an almost negligible amount of gamma-globin in SF culture stimulated by 100 U/mL interleukin-3 (IL-3) and 2 U/mL Ep, while corresponding FCS culture revealed significant gamma-globin biosynthesis. The %FNRBC of the erythroid bursts in SF cultures derived from nine normal adults (2.0% +/- 0.9% F cells) was 3.0% +/- 3.4%, while in FCS culture, it was 25% +/- 12% (mean +/- SD). Dosages of IL-3 between 10 and 10,000 U/mL did not increase %FNRBC in FCS of SF conditions. Mean Hb contents of nucleated erythrocytes (NRBC) assayed by microdensitometry of pericellular immunoprecipitate were similar in FCS and SF cultures. The number of erythroid bursts per 2 x 10(3) CD34-positive marrow cells was 48 +/- 20 in FCS and 36 +/- 12 in SF cultures. In two experiments, progenitors grown for 7 days under SF conditions were isolated and recultured in either SF or FCS conditions for 7 days, and the resulting erythroid bursts were analyzed for FNRBC. The bursts that had been returned to FCS cultures yielded values of %FNRBC intermediate between those obtained from progenitors grown entirely in SF or FCS cultures, indicating that serum effect is not solely due to growth selection for certain subpopulations of erythroid burst-forming units. This experiment also demonstrated that the factors present in serum responsible for HbF augmentation act at both early and late stages during erythroid burst development. SF culture of peripheral blood progenitors of one subject with heterocellular hereditary persistence of fetal hemoglobin (HPFH) yielded elevated levels of FNRBC (19% +/- 5%) that accurately reflected the F cell (18%) of the circulating blood. Similarly, FNRBC in cultures of progenitors from one umbilical cord blood sample (86% F reticulocytes) was 87 +/- 3% FNRBC. The SF culture for enriched human progenitors, which nearly reflects the physiologic HbF programs of the donor, should facilitate studies of the exact mechanisms of postnatal reactivation of HbF production.