Fractionation of subsets of BFU-E from normal human bone marrow: responsiveness to erythropoietin, human placental-conditioned medium, or granulocyte-macrophage colony-stimulating factor

Normal human bone marrow mononuclear cells were fractionated by differential adherence, immunomagnetic separation, and fluorescence- activated cell sorting (FACS). The resultant fractionated cells were cultured in semisolid medium to monitor the presence of BFU-E, Mix-CFC, and nonerythroid CFC. Two populations of cells were recovered on the basis of binding by the monoclonal antibody (MoAb) RFB-1. One of these populations contained BFU-E that were stimulated only by erythropoietin (Epo), whereas the second population contained BFU-E responsive to Epo, Epo and recombinant human granulocyte-macrophage colony-stimulating factor (rHGM-CSF), or Epo and human placental-conditioned medium (HPCM). Prior enrichment of clonogenic cells by removal of adherent and Leu-M3+ve, Leu-4+ve, Leu-7+ve, B1+ve, WEMG1+ve, and Glycophorin A+ve cells, followed by FACS fractionation on the basis of RFB-1 binding, consistently resulted in recoveries of BFU-E, Mix-CFC, and nonerythroid CFC of greater than 100% (up to 800%). These procedures also resulted in enrichment of up to 200-fold and frequencies of 1:6 for BFU-E, 1:5 for CFC, and 1:130 for Mix-CFC.     

Expression of CD11, CDw15, and transferrin receptor antigens on human hematopoietic progenitor cells

The expression of transferrin receptor-associated antigens and of CD11 and CDw15 antigens was investigated on myeloid committed progenitor cells (CFU-GM day 10, CFU-GM day 7, and cluster-forming cells [CFC] day 4), on erythroid committed progenitor cells (BFU-E and CFU-E), and on multilineage progenitor cells (CFU-GEMM). Both complement-dependent cytotoxicity and fluorescence-activated cell-sorting assays were performed. Complement-dependent cytotoxicity appeared to be the more sensitive assay. Transferrin receptor-associated antigens appeared to be clearly present on all myeloid and erythroid committed progenitor cells, but were found to be only weakly expressed on CFU-GEMM. CD11 antigens appeared to be strongly expressed only on mature granulocytes, monocytes, and certain lymphocytes, but not significantly on myeloid committed precursor cells. Surprisingly, CD11 antigens were weakly, but significantly, present on CFU-E. CDw15 antigens appeared to be restricted to myeloid differentiation and were increasingly expressed from CFU-GM day 10 to CFC day 4. Thus, antitransferrin receptor, CD11, and CDw15 antibodies can be used to separate hematopoietic progenitor cells and may be useful tools in the study of hematopoietic differentiation.     

Expression of latent hematopoietic progenitor cells in cultures of newborn and adult baboon liver

The anatomic site of hematopoiesis changes during fetal development from the yolk sac to the liver and finally to the marrow. Factors controlling this switch in the site of hematopoiesis are unknown. We assayed erythroid colony (CFU-E) and erythroid burst (BFU-E) formation in fetal, newborn, and adult baboon liver and marrow to determine the growth requirements of primate hematopoietic progenitor cells from different anatomic sites and developmental stages. We cocultured fetal, newborn, and adult liver and marrow nonadherent cells with adherent cells from these organs to assess the role adherent cells may play in determining the site of hematopoiesis. Fetal liver, fetal marrow, newborn marrow, and adult marrow cultures formed CFU-E and BFU-E colonies in vitro. In contrast, newborn and adult liver cell cultures very rarely formed colonies. However, when newborn or adult liver nonadherent cells were cocultured with marrow adherent cells, CFU-E and BFU-E colonies were detected. The colonies that formed in the newborn and adult liver cultures were derived from the liver and not from the marrow cells or peripheral blood trapped in the liver. These data suggest that in contrast to fetal liver, newborn and adult liver may not be hematopoietic organs in normal primates in vivo because of changes in the growth requirements of hematopoietic progenitor cells present in these organs.     

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.     

Sources and nature of granulocyte-macrophage colony stimulating factor in fetal mice.

At the earliest stages of fetal hepatic hemopoiesis in CBA mice (11-12 days gestation), colony stimulating activity could be found only in peripheral blood, yolk-sac fluid and media conditioned by yolk-sacs (YSCM). The colony stimulating factor (GM-CSF) from YSCM was able to be concentrated by absorption to DEAE-cellulose and subsequent elution. Titration of this material produced a sigmoid dose-response curve in agar cultures of adult CBA bone marrow cells. Unlike the high proportion of granulocyte colonies stimulated by the GM-CSF from mouse lung conditioned medium, all concentrations of YSCM produced a high proportion of macrophage colonies after 7 days of incubation. Mixing experiments eliminated the possibility that a specific inhibitor preventing granulocyte differentiation was present in YSCM. Fetal liver cells were relatively unresponsive to YSCM, but their ability to respond increased with gestational age. When stimulated by YSCM, fetal liver colony forming cells from mice of all gestational ages produced more than 90% macrophage colonies after 7 days of incubation. The experimental data suggest that the proliferation and differentiation of granulocyte and macrophage precursors in the early fetal liver could be controlled by a fetal type of GM-CSF favoring macrophage production.     

Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3

Mice lacking suppressor of cytokine signaling 3 (SOCS3) exhibited embryonic lethality with death occurring between days 11 and 13 of gestation. At this stage, SOCS3(-/-) embryos were slightly smaller than wild type but appeared otherwise normal, and histological analysis failed to detect any anatomical abnormalities responsible for the lethal phenotype. Rather, in all SOCS3(-/-) embryos examined, defects were evident in placental development that would account for their developmental arrest and death. The placental spongiotrophoblast layer was significantly reduced and accompanied by increased numbers of giant trophoblast cells. Delayed branching of the chorioallantois was evident, and, although embryonic blood vessels were present in the labyrinthine layer of SOCS3(-/-) placentas, the network of embryonic vessels and maternal sinuses was poorly developed. Yolk sac erythropoiesis was normal, and, although the SOCS3(-/-) fetal liver was small at day 12.5 of gestation (E12.5), normal frequencies of erythroblasts and hematopoietic progenitor cells, including blast forming unit-erythroid (BFU-E) and, colony forming unit-erythroid (CFU-E) were present at both E11.5 and E12.5. Colony formation for both BFU-E and CFU-E from SOCS3(-/-) mice displayed wild-type quantitative responsiveness to erythropoietin (EPO), in the presence or absence of IL-3 or stem cell factor (SCF). These data suggest that SOCS3 is required for placental development but dispensable for normal hematopoiesis in the mouse embryo.     

Fractionation of normal and beta-thalassemic human hemopoietic progenitor cells by immunomagnetic beads

Depletion of adherent cells, followed by simultaneous immunomagnetic bead depletion of Leu 4+, Leu 7+, Leu 11+, Leu M1+, Leu M3+, B1+, WEM-G11+, and Glycophorin A+ cells from normal bone marrow mononuclear cells, consistently led to recoveries of erythroid and nonerythroid colony-forming cells of greater than 100% and enrichment of 13- to 99-fold. Similarly, the recovery of mixed erythroid colony-forming cells was greater than 100%, with enrichments of 7.5- to 262-fold. This simple procedure, when used on normal bone marrow and beta-thalassemic peripheral blood mononuclear cells, as well as providing significant enrichment, suggests that colony assays on unfractionated human mononuclear cells specifically underestimate the number of BFU-E, Mix-CFC, and nonerythroid-CFC present.     

Fetal erythropoiesis in steel mutant mice. III. Defect in differentiation from BFU-E to CFU-E during early development

Erythroid progenitor cells in +/+ and Sl/Sld fetal livers manifested as burst-forming units-erythroid (BFU-E) and colony-forming units- erythroid (CFU-E) were assayed in vitro during early development. The proportion of BFU-E was higher as mutant than in normal fetal livers. On the other hand, the proportion of CFU-E was less in the mutant than in the normal. These results suggest that the defect in Sl/Sld fetal hepatic erythropoiesis is expressed at the steps of differentiation that effect the transition from BFU-E to CFU-E.     

Characterization of hemopoietic activities in media conditioned by a murine marrow-derived adherent cell line, B.Ad

The hemopoietic activities present in medium conditioned by a murine bone marrow-derived adherent cell line (B.Ad) have been studied. B.Ad-conditioned medium stimulated neutrophil, neutrophil-macrophage, and macrophage colonies in agar cultures of bone marrow cells and 90% of this activity was neutralized by antimacrophage colony-stimulating factor (anti-M-CSF). The conditioned medium supported the generation and/or maintenance of spleen colony-forming units (CFU-S) in liquid cultures and synergized with multilineage colony-stimulating factor (Multi-CSF; IL-3) to stimulate colony formation by day-3 post-5-fluorouracil (FU)-treated bone marrow cells. When used as feeder layers, B.Ad cells stimulated erythroid colony-forming units (CFU-E) and markedly enhanced erythroid burst-forming units (BFU-E) stimulation more than did maximal Multi-CSF (IL-3) and Epo stimulation. No CFU-E- or BFU-E-stimulating activities were detected in medium conditioned by B.Ad cells. Similarly, B.Ad-conditioned medium was unable to stimulate Multi-CSF (IL-3) or granulocyte-macrophage (GM)-CSF-dependent cell lines. The data suggest that medium conditioned by this bone marrow-derived adherent cell line contains M-CSF and other factors not detectable as CSFs that either directly or by means of a synergistic mechanism are able to stimulate CFU-S and colony-forming cells (CFC).     

In vitro steroid sensitivity testing: A possible means to predict response to therapy in primary hypoproliferative anemia

We investigated the effects of various steroids on erythroid colony formation by normal human bone marrow and peripheral blood, and by marrow and peripheral blood from 18 patients with primary hypoproliferative anemia. These agents were variously found to enhance both CFU-E and BFU-E derived colony growth by normal human cells. Fluoxymesterone and dexamethasone were the most active inducers of CFU-E proliferation, and etiocholanolone and dexamethasone were the most potent burst augmenters. Androsterone did not significantly influence BFU-E proliferation in 66% of the marrow cultures from hematologically normal donors. Colony formation by erythroid progenitor cells of the patients with hypoproliferative anemia was reduce (20 ± 10 CFU-E derived colonies/6 × 10⁴ marrow cells; 12 ± 5 BFU-E derived colonies/1 × 10⁵ blood cells) when compared to growth by normal cells (65 ± 14 CFU-E derived colonies/6 × 10⁴ marrow cells; 21 ± 9 BFU-E derived colonies/1 × 10⁵ blood cells). Colony formation by marrow or peripheral blood cells of eight patients with steroid-responsive anemia was only moderately reduced (26 ± 11 CFU-E derived colonies/6 ± 10⁴ marrow cells; 17 ± 3 BFU-E derived colonies/1 × 10⁵ blood cells) when compared to growth by marrow cells of three steroid-unresponsive patients (3 ± 1.5 CFU-E derived colonies/6 × 10⁴ cells). Whereas the addition of steroids of the same class to marrow and peripheral blood cultures of the steroid-responsive patients enhanced colony growth by 60–300%, their addition to marrow cultures of the steroid-unresponsive patients increased colony growth by less than 60%. It appears that further investigations using in vitro culture techniques as predictors of response to steroid therapy in patients with hypoproliferative anemia may be warranted.     

Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast

Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.     

Changes in expression of major histocompatibility complex (MHC) class-I antigen on hematopoietic progenitors during murine development

Changes in the expression of H-2K antigen on murine hematopoietic progenitors (CFU-E, BFU-E, CFU-C and CFU-mix) were examined during murine development using the complement-dependent cytotoxicity assay. The results revealed that CFU-mix, BFU-E, and CFU-E had begun to express H-2K antigen as early as day 16 of gestation, and antigens increased sequentially thereafter. On the other hand, CFU-C expression began as late as days 12-15 after birth and the antigen increased rapidly until week 8 after birth. Expression of H-2K antigen on CFU-mix was higher than that on CFU-E and BFU-E, while that on CFU-C was lowest during the period between day 16 of gestation and days 12-15 after birth. The possibility of an indirect effect of antibodies on T cells was unlikely because thymocytes added to week-8 bone marrow cells after antibody treatment did not produce a significantly different effect on the culture.     

Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (TÜ 67)

Summary Anti-TÜ 67 is a murine monoclonal antibody that recognizes the transferrin receptor. With respect to hematopoietic cells TÜ 67 is expressed by human multipotent colony-forming cells (CFU-Mix), erythroid progenitor cells (BFU-E and CFU-E) and a fraction of granulocyte/monocyte colony forming cells, but is not expressed by mature hematopoietic cells including erythrocytes, platelets, lymphocytes, and peripheral blood myeloid cells. The TÜ 67-positive fraction of normal bone marrow, separated by fluorescence-activated cell sorting (FACS) or immune rosettes, contained 87% of the erythroid progenitor cells. Erythroid progenitor cells were enriched up to 50-fold by using a combination of monoclonal antibodies to deplete mature hematopoietic cells, followed by positive selection of BFU-E and CFU-E by TÜ 67 antibody.Supported by the Deutsche Forschungsgemeinschaft (He 1380-2/1)     

Erythropoietin receptor characteristics on primary human erythroid cells.

Erythropoietin (EP) exerts its effects on erythropoiesis by binding to a cell surface receptor. We examined EP receptor expression during normal human erythroid differentiation and maturation from the burst-forming unit-erythroid (BFU-E) to the reticulocyte level. In contrast to previous studies, we assessed EP receptor number and affinity in erythroid precursors immunologically purified from fresh bone marrow aspirates or fetal liver samples and in reticulocytes purified from peripheral blood. EP receptors were quantitated by equilibrium binding experiments with 125I EP. We found that purified primary erythroblasts from both adult and fetal sources exhibited a single high-affinity (kd 100 pmol/L) binding site for EP under our experimental conditions, and 135 or 250 receptors per cell, respectively. Reticulocytes were devoid of EP receptors. We compared these data to in vitro-derived BFU-E progeny at both early and late stages of maturation. Cultured BFU-E progeny also displayed a single class of receptors of slightly lower affinity (210 to 220 pmol/L). Preparations enriched in colony-forming units-erythroid (CFU-E) and proerythroblasts (day 9 BFU-E progeny) displayed approximately 1,100 receptors per cell, whereas populations containing mature erythroblasts (day 14 BFU-E progeny) exhibited approximately 300 receptors per cell. Furthermore, information from binding experiments was complemented by autoradiography in both enriched BFU-E preparations, cultured BFU-E progeny (days 9 and 14), and marrow mononuclear cells. These studies are consistent with a peak in EP receptor expression at the CFU-E/proerythroblast stage and a decrease with further maturation to undetectable levels at the reticulocyte stage. These data examining EP receptor characteristics on freshly isolated erythroid precursor cells complement previous data on EP receptor biology using culture-derived erythroblasts.     

The influence of benzene on the erythroid cell system in mice

Female BDF1 mice were exposed up to 8 weeks to airborne concentrations of 100, 300, and 900 ppm of benzene, 6 h/day, 5 days/week. The erythropoietic cell compartment in the bone marrow and the peripheral blood was studied using the erythroid burst-forming unit (BFU-E) and erythroid colony-forming unit (CFU-E) assays, the incorporation of 59Fe, and standard methods. In the peripheral blood only a slight anemia was observed. In the bone marrow, however, a considerable decrease of CFU-E numbers was seen, the CFU-E being more depressed than the BFU-E numbers. In bone marrow smears a variable content of erythroblasts was found. The 59Fe kinetics showed an enhanced turnover within the erythron, suggesting the decrease in transit time as a compensating mechanism for the low CFU-E numbers. After 4 weeks of exposure to all benzene concentrations, greater than 17 days in benzene-free atmosphere are needed for a complete recovery of BFU-E and CFU-E compartment sizes.     

Mechanism of extramedullary haematopoiesis in rabbits with saponin-induced myelofibrosis and myeloid metaplasia

S ummary . The saponin-induced myelofibrosis and myeloid metaplasia model in the rabbit was used to study mechanisms of extramedullary haematopoiesis. Haematopoietic progenitor cells, erythroid colony forming units (CFU-E) and burst forming units (BFU-E) were assayed serially in the peripheral blood, spleen and bone marrow after saponin administration, employing the in vitro , methylcellulose culture technique. Animals that had undergone splenectomy prior to saponin administration were also studied. The results demonstrated increases of progenitor cells in the blood and spleen and a simultaneous depletion of such cells in marrow after saponin treatment. The results in splenectomized animals were similar to those observed in non-splenectomized animals after saponin administration. The findings indicate that following saponin administration there is a release of CFU-E and BFU-E from bone marrow into periphery and probably deposition in the spleen, and suggest that myeloid metaplasia in myelofibrosis may result from colonization of extramedullary sites originating from the bone marrow.     

Kinetics of erythroid precursors in mice infected with the anemic or the polycythemic strain of Friend leukemia virus

The kinetics of both erythroid burst-forming and colony-forming units (BFU-E, CFU-E) and myelomonocytic precursors [myelomacrophage colony-forming unit (CFU-C)] have been evaluated in tibial marrow, peripheral blood, and spleen of DBA/2 mice at time intervals after inoculation of either the anemic (FLV-A) or the polycythemic (FLV-P) strain of Friend leukemia virus. Either one of the viruses induced, at 7-10 days after infection, a massive increase in the number of BFU-Es in peripheral blood, in parallel with their depletion in tibial marrow and increase in spleen. A comparable increase in the blood BFU-E number was observed in splenectomized FLV-infected mice. These results indicate a marrow-spleen migration of BFU-Es. In spleen, the increase of the BFU-E number was associated with an increase in the CFU-E pool. In tibial marrow, a sequence of expansion/depletion waves occurred reciprocally at the level of BFU-E and CFU-E. The cycling of BFU-E([³H]thymidine in vitro suicide index) in marrow, blood, and spleen was enhanced, whereas that of CFU-E and CFU-C showed little or no modification. These kinetic data suggest that the main target cell of FLV may be the BFU-E or a closely related element. In plates without added erythropoietin (but containing it in fetal calf serum), expression of CFU-E from FLV-P-treated animals was maximal; that of CFU-E from FLV-A-injected mice was either virtually absent or only slight in marrow or spleen, respectively. BFU-E growth always was fully dependent upon erythropoietin addition. Control studies in FLV-infected resistant mice and in susceptible mice given diluted or heat-inactivated virus provide convincing evidence that the phenomena described are induced by FLV.     

SFL 23.6: a monoclonal antibody reactive with CFU-E, erythroblasts, and erythrocytes

A cytotoxic (IgG2b) monoclonal antibody (McAb) for a novel erythroid differentiation antigen was generated by hyperimmunizing young mice with mononuclear cells obtained from livers of 20- to 22-week-old fetuses. This McAb, designated SFL 23.6, shows an extremely well- defined reactivity with the cells of the erythroid lineage at all stages of maturation as evident from the labeling of morphologically identifiable erythroid precursors and of erythrocytes present in peripheral blood, bone marrow, and fetal liver, and from its reactivity with culture-derived erythroblasts. The nonerythroid cells present in these and other tissue preparations were not labeled by SFL 23.6. The erythroid lineage specificity of McAb SFL 23.6 was confirmed by a cell- sorting experiment in which 97% of the cells in the fluorescent fraction sorted from SFL 23.6-treated bone marrow cells were erythroid precursors at various stages of maturation. Complement-mediated cytotoxicity and progenitor cell-sorting experiments showed that most (greater than 90%) of the late erythroid progenitors (CFU-E) and only a small proportion of the early erythroid progenitors (BFU-E) express the antigenic determinant identified by SFL 23.6. The myeloid progenitors (CFU-GM) and multilineage progenitors (CFU-GEMM) were negative for the SFL 23.6 antigenic determinant. The antigen recognized by SFL 23.6 has not been determined as yet. Because of the pattern of its reactivity and its dependence on sialic acid residues, the possibility of its relationship to glycophoria A was entertained. However, previous work using antiglycophorin McAbs (R-10) has shown that this determinant is not expressed in CFU-E. Therefore, among the erythroid lineage-specific McAbs described thus far, SFL 23.6 is unique in its reactivity with CFU- E and the mature erythron. Reagents with such specificity may be useful in studies of erythroid differentiation and commitment.     

Development of pluripotent hematopoietic progenitor cells in the human fetus

Pluripotent hematopoietic progenitor cells (CFU-GEMM), myeloid progenitor cells (CFU-GM), and erythroid progenitors (BFU-E) were studied in midtrimester human fetuses using the mixed colony assay. All three progenitor cell populations were detected at high levels in the fetal liver from 12 to 23 wk of gestation. Stem cells were first observed in the bone marrow at 15-16 wk of gestation, although bone marrow cultures from earlier fetuses showed heavy growths of stromal cells. Spleen cultures first showed growth of stem cells at 18-19 wk, but fetal thymus showed no hematopoietic activity. Peripheral blood from four fetuses aged 13, 18, 20, and 21 wk showed very high levels of all 3 progenitor cells. The results demonstrate that hematopoietic development in the human fetus parallels that of the mouse. The observation that stromal cell development in the bone marrow precedes the appearance of hematopoietic progenitor cells suggests that they may be closely involved in stem cell growth.