Megakaryocyte colony-stimulating factor and burst-promoting activity in LPS-treated mouse spleen cell-conditioned medium

In order to clarify the mechanism(s) of increased splenic hematopoiesis noted in lipopolysaccharide (LPS)-injected mice, the effects of spleen cell-conditioned medium (SPCM) on megakaryocyte colony (CFU-meg) formation and early erythroid (BFU-e) differentiation were investigated. After spleen cells from LPS-injected mice were incubated for 3 days, the SPCM was assayed for megakaryocyte colony-stimulating factor (Meg-CSF) in CFU-meg assay and for burst-promoting activity (BPA) and erythropoietin (Epo) in erythroid colony assays (i.e., CFU-e, BFU-e). Colony formation of CFU-meg and BFU-e peaked with the addition of 30 and 10-15% SPCM, respectively. Spleen cells from LPS-injected mice produced Meg-CSF and BPA when compared with controls. However, conditioned medium from spleen cells depleted of phagocytic cells had low Meg-CSF and BPA. SPCM did not contain detectable quantities of Epo. It appears likely that local splenic production of Meg-CSF and BPA may affect proliferation of CFU-meg and erythroid progenitor cells in the spleen.     

Comparative distribution of marrow CFU-e and CFU-gm progenitors in different anatomic sites in the dog

The interpretation of marrow cloning activity, particularly in serial cultures, is greatly influenced by the reproducibility of the collected marrow samples. In order to establish whether bone marrow cloning activities and precision of the cloning assays are influenced by the site of bone marrow collection in the dog, we studied the incidence of marrow erythroid (CFU-e) and granulocyte-macrophage (CFU-gm) progenitor cells in the iliac crest, sternum, vertebrae, femur, and humerus, using microplasma clot and soft agar culture systems. Marrow samples obtained from the femur and humerus revealed consistently higher cell concentrations than those from the iliac crest, vertebrae, or sternum. Those aspirated from the sternum and vertebrae had lower cell concentrations and were less reproducible. Statistical analysis revealed no significant differences in the incidence of marrow CFU-e and CFU-gm progenitor cells between the femur, humerus, iliac crest or vertebrae. With multiple sampling, the marrow cloning efficiency was consistent and reproducible within the individual dogs. We conclude that the distribution of CFU-e and CFU-gm is comparable throughout the active marrow in the dog and that these sites may be used interchangeably for multiple quantitative analysis of marrow hematopoietic progenitor cells.     

Effects of recombinant human tumor necrosis factor (rhTNF) on normal human and mouse hemopoietic progenitor cells

We examined the effects of recombinant human tumor necrosis factor (rhTNF) on normal human and murine granulocyte-macrophage (CFU-gm) and erythroid (CFU-e, BFU-e) progenitor cells. We suppressed in vitro colony formation by human marrow CFU-gm, CFU-e and BFU-e or peripheral blood BFU-e by adding rhTNF to the culture in a dose-related manner. A half-maximal inhibition was observed with 1-10 ng/ml. Leukemic cell line K562 cells were found to be sensitive to rhTNF in the clonogenic colony assay. However, the clonal growth of murine marrow CFU-e and BFU-e colonies was less than 50% inhibited and CFU-gm growth was unaffected even at a concentration of 1,000 ng/ml. We observed slight to moderate inhibition after 24 h pulse exposure of both human and murine-committed progenitors to rhTNF prior to the culture. Intravenous injection of 1 mg/kg of rhTNF caused a marked decrease in marrow erythroid progenitors and consequently caused anemia in the mice. Our data indicate that rhTNF has a suppressive effect on normal human and murine hemopoietic colony formation in vitro and murine erythropoiesis in vivo.     

Growth of erythroid colonies in agar cultures of normal human bone marrow

The use of methylcellulose (MC) gels or plasma clots, for the support of human erythropoiesis in vitro, is associated with several technical disadvantages. Substitution of soft agar offers the prospect of overcoming these difficulties. In comparative studies, normal human bone marrow cells were cultured with erythropoietin (Epo) in agar (0.1%-0.3%) and MC. Concentrations of 0.175% and 0.2% agar proved to be optimal with respect to the combination of cloning efficiency and colony density. Further morphological examination revealed that subcolony formation in erythroid 'bursts' was influenced by gel viscosity. In additional experiments, miniaturising the assay system, to 0.25 ml culture volumes, increased cloning efficiency and reduced Epo utilization. These results confirm and expand earlier observations, and support a preference for the general use of agar in human erythroid cell cultures.     

Effect of recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on human BFU-e in serum-free cultures

The effects of recombinant human hemopoietic growth factors on early and late human erythroid progenitors (BFU-e and CFU-e) were investigated in serum-free cultures. Recombinant human erythropoietin (rhEpo) induced the formation of not only human CFU-e-derived colonies but also human BFU-e-derived bursts. Recombinant human interleukin 3 (rhIL-3) alone did not induce the formation of human BFU-e-derived bursts and human CFU-e-derived colonies. In the presence of rhEpo, rhIL-3 dose dependently increased the number of bursts stimulated by rhEpo, although rhIL-3 did not have the augmentative effect on human CFU-e growth. On the other hand, rhIL-3 did not stimulate the formation of murine BFU-e-derived bursts, and murine IL-3 did not stimulate the formation of human BFU-e-derived bursts. The results indicated that the burst-promoting activity of IL-3 was species-specific between human and murine cells. Recombinant human GM-CSF (rhGM-CSF) or recombinant human G-CSF (rhG-CSF) failed to induce human burst formation and did not augment the effect of rhEpo on human burst formation. The results of the present study suggest that in vitro, IL-3 can stimulate BFU-e in collaboration with Epo, but GM-CSF and G-CSF do not stimulate BFU-e growth in the presence or absence of Epo.     

Effects of increasing cyclic AMP or calcium on feline erythroid progenitors in vitro: normal cells are stimulated while cells from retrovirus-infected cats are suppressed

Pharmacologic modulators of cyclic 3',5'-adenosine monophosphate (cAMP) and calcium were added to cultures of bone marrow cells from normal cats and cats with retrovirus-induced erythroid aplasia (EA). Treatment with the following reagents increased the number of erythroid progenitors (CFU-e and BFU-e) in cells from normal cats: isoproterenol, dibutyryl cAMP, forskolin, RO-20-1724 and A23187. However, treatment of cells collected from viremic cats not only failed to enhance CFU-e and BFU-e but inhibited their growth. These studies suggest that EA is related to a non-reversible block of primitive erythroid progenitors or to direct inhibition of BFU-e and CFU-e growth.     

Differential effects of human erythroid burst stimulating activity (HuEBSA) on human cord blood burst forming units-erythroid (BFU-Es) as a function of their differentiation state.

An erythroid stimulating activity which promotes the growth of small bursts probably arising from mature burst forming units-erythroid (BFU-Es) of adult human bone marrow cells and called human erythroid burst stimulating activity (HuEBSA), was previously found in media conditioned by a fetal human kidney cell line. In the present work we report that adding HuEBSA to cultures did not increase the burst number but increased the size of bursts from cord blood (CB) cells. A similar observation was made using stem cell factor (SCF). However, a synergistic effect on the burst number was noted when both HuEBSA and SCF were introduced to cultures. We also noticed that CB erythroid progenitors pre-cultured with 5637-Conditioned Medium [as a source of burst promoting activity (BPA)] and erythopoietin (Epo) for 3 days could be stimulated by HuEBSA but not by SCF. Similar results were obtained when interleukin 3 (IL-3) was introduced with Epo to the pre-cultures. These results suggest that two different populations of erythroid progenitors coexist in cord blood, one is Epo- and IL-3-sensitive, the other solely Epo-sensitive. It also seems probable that HuEBSA acts on erythroid progenitors arising from the more immature erythroid population, since its stimulating activity was evident after a 3-day pre-culture of cord blood cells in Epo and IL-3.     

Use of gel-well culture chambers as a liquid culture system to measure responses of hematopoietic colony-forming cells to growth factors

This report presents the results of an investigation in which Gel-Well culture chambers were evaluated for their utility as a liquid culture assay system to measure the responses of hematopoietic colony-forming cells (CFC) to recombinant and cell-derived growth factors. Gel-Wells, designed for anchorage-independent cell growth and diffusion of media components, permitted the weekly replacement of media and growth factors without removing cells from the culture chambers. In these studies, changes in cellularity and CFC content in Gel-Well cultures of human umbilical cord blood cells induced by recombinant interleukin 3 (rIL-3) were quantified. After one week in culture without rIL-3, the number of erythroid burst-forming units (BFU-e) had decreased to 25 +/- 38% of pre-values. In contrast, addition of rIL-3 induced an increase in the number of BFU-e to 390 +/- 135% of pre-values. By three weeks with rIL-3, the number of granulocyte-macrophage colony-forming units (CFU-gm) had increased to 292 +/- 58% of pre-values. Also, the presence of a bone marrow stromal cell layer under the Gel-Well helped to maintain the survival of CFC in liquid culture. These studies demonstrated that Gel-Well culture chambers provide a useful liquid culture system for studying the responses of CFC to growth factors.     

Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells

The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells. The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively. Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples. The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells. This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.     

Erythropoietic progenitor cells from premature neonates studied using a validated serum-deprived medium

We have examined a serum-deprived culture system in order to verify that it is suitable for the study of burst forming unit erythroid (BFU-E) progenitor cells from premature neonates. Optimum growth of BFU-E from premature neonates was observed with each media constituent using the same concentration as that previously described for adult subjects. Growth of immature BFU-E from premature neonates were highly dependant upon a source of Burst Promoting Activity and mature BFU-E derived colonies emerged at day 12 compared to day 14 in adults. Our preliminary results with the validated medium suggest that premature infants have increased peripheral blood concentrations of BFU-E compared to healthy adult controls.Abbreviations Ad Adherent cells - BPA Burst promoting activity - BFU-E Burst forming unit erythroid - Epo Erythropoietin - IL3 Interleukin-3 - LDC Low density (<1.077 g ml¹) peripheral blood mononuclear cells     

Growth of human normal erythroid progenitors in liquid culture: a comparison with colony growth in semisolid culture

Most studies of erythropoiesis in vitro have employed cloning methods in semisolid medium. We have recently described a two-step liquid culture procedure that supports the proliferation and differentiation of human erythroid progenitors. In the present study, we have modified the procedure to allow large-scale cultures of erythroid cells derived from normal donors. The culture is divided into two phases. In the first phase, which is erythropoietin (Epo) independent, the early erythroid progenitors multiply and differentiate. In the second, Epo-dependent phase, they mature into orthochromatic normoblasts and enucleated erythrocytes. Using this procedure, erythroid cell yield reached 7.5 x 10(6)/ml and a total of 7 x 10(8) cells could be harvested per blood unit. A comparison of the growth of erythroid cells in liquid culture to their colony growth in semisolid culture indicated that cell growth was superior: 1) in liquid culture in terms of cell yield per originally cultured mononuclear cell, 2) per ml culture and per culture surface area and in the purity of the resultant erythroid cell population. In addition, it permits easier manipulation of the culture condition and components and sampling of greater than 1 x 10(7) cells at each maturation stage subsequent to the proerythroblast stage. This liquid culture procedure might provide an important experimental tool for studying erythroid cell development.     

Effects of recombinant human erythropoietin on hematopoietic progenitors of chronic hemodialysis patients in vitro and in vivo

Erythroid progenitors were assayed in 20 chronic hemodialysis patients before and after administration of recombinant human erythropoietin (rhEpo) using a methylcellulose culture method. Hemoglobin concentrations, hematocrit values and the proportion of marrow erythroblasts increased significantly during rhEpo treatment. The numbers of erythroid progenitors (CFU-e and BFU-e) in the patients' marrow also increased following rhEpo administration. Our data suggest that rhEpo is an effective drug for treating anemia caused by chronic renal failure and the administration of rhEpo results in an increase in the numbers of erythroid progenitors.     

Effects of five recombinant hematopoietic growth factors on enriched human erythroid progenitors in serum-replaced cultures

Erythroid progenitors from normal human marrow were purified by a two-step immune panning method permitting both the enrichment of erythroid progenitors (plating efficiency up to 10%) and the separation of CFU-E from BFU-E. The purified erythroid progenitors were grown in serum-replaced conditions; in some experiments at an average of one cell per well. Human recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL3), erythroid potentiating activity (EPA), and human erythropoietin (Epo) either recombinant or homogenous native were tested for their effect on CFU-E growth. Epo was an absolute requirement for CFU-E growth and was sufficient to obtain colony formation at the unicellular level whereas GM-CSF and IL3 did not further increase the plating efficiency. EPA potentiated the effect of Epo on this progenitor only in experiments performed at unicellular level. Human recombinant GM-CSF, IL3, Interleukin 1 alpha (IL1 alpha), and Epo were subsequently tested for their ability to promote BFU-E growth. GM-CSF and IL3 supported the growth of erythroid bursts in the presence of Epo, even at the unicellular level. However, IL3 promoted a higher number of bursts than GM-CSF under all conditions tested. These two growth factors have no or very small additive effects when tested in combination. IL1 alpha added to Epo alone had no effect on the growth of BFU-E whereas it potentiated the combined action of IL3 and GM-CSF on the primitive BFU-E. In conclusion, this study confirms at the unicellular level and under serum-free conditions that erythroid progenitors are regulated by multipotential growth factors in early phases of erythropoiesis and become sensitive only to Epo in later phases of differentiation.     

Direct effects of IL-4 on the in vitro differentiation and proliferation of hematopoietic progenitor cells

The purpose of this study was to analyze the effects of recombinant human interleukin 4 (IL-4) on the differentiation and proliferation in vitro of human granulocyte/macrophage (GM) and erythroid progenitors. IL-4 was added to either fetal bovine serum (FBS)-supplemented or to FBS-deprived cultures of unfractionated human marrow cells or marrow cells depleted of adherent and/or T cells. Paradoxical effects similar to those reported in the murine system were detected in these experiments. In FBS-supplemented cultures, IL-4, which had no effect on the growth or erythroid bursts (from burst-forming cells; BFU-E) detected in the presence of Epo alone, decreased by 46% the number of erythroid bursts detected in the presence of Epo and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). In contrast, in FBS-deprived cultures, IL-4 increased by 30-700% the number of erythroid bursts in cultures containing Epo alone or containing Epo, IL-3, and GM-CSF. The stimulatory effect of IL-4 on erythroid burst growth under FBS-deprived conditions was particularly evident when adherent cells were removed. Under the conditions investigated, IL-4 had little effect on the growth of GM colonies. In FBS-deprived suspension cultures of nonadherent, T-cell-depleted marrow cells, IL-4 maintained both the number of BFU-E and CFU-GM for at least 8 days. In these cultures, IL-4 antagonized the capacity of IL-3 to increase the number of BFU-E but IL-4 and IL-3 acted together to maintain the number of CFU-GM. To determine if IL-4 acted directly or indirectly, its effects on the growth of factor-dependent subclones of the murine progenitor cell line 32D were analyzed. Three subclones were studied: the original IL-3-dependent clone 32D cl.3, the Epo-dependent erythroid clone 32D Epo-1, and the G-CSF-dependent myeloid clone 32D G-1. IL-4 alone failed to induce colony growth from these cell lines. However, IL-4 inhibited by 25% the number of colonies formed by 32D cl.3 in the presence of IL-3 while increasing by 25% and 25-50% the number of colonies formed by 32D Epo-1 and 32D G-1 in the presence of Epo or G-CSF, respectively. These results indicate that human IL-4, as its murine counterpart, is a multilineage growth factor with paradoxical effects which are mediated by the direct action of IL-4 on progenitor cells.     

Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the nasa rotating wall vessel bioreactor

Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.     

Burst-promoting activity in anemia and polycythemia

Burst-promoting activity (BPA) in the sera of patients with various types of anemia and polycythemia was compared with that of normal subjects by an in vitro method using mouse bone marrow cells. The control culture contained normal human AB serum instead of sample materials. Results were expressed as a percentage of burst numbers in control cultures. Serum erythropoietin (Epo) levels were determined by a radioimmunoassay. Serum BPA in patients with aplastic anemia (155.4 +/- 56.7%, mean +/- SD) was significantly higher than that in normal subjects (112.1 +/- 29.1%, Wilcoxon's rank sum test, P less than 0.05). However, serum BPA in patients with uremic anemia (122.2 +/- 26.5%), polycythemia vera (101.9 +/- 19.5%) and stress polycythemia (115.5 +/- 25.6%) was not significantly different from normal subjects. There was a correlation between serum BPA and Epo titers in patients with aplastic anemia and paroxysmal nocturnal hemoglobinuria (r = 0.81, t test, P less than 0.001).     

Velocity sedimentation profiles of normal and regenerating primitive erythroid burst-forming units: relation to phase of cell cycle and growth in vitro

Abstract. The primitive burst-forming unit-erythroid (BFU-e) derived from normal and regenerating murine bone marrow was examined by velocity sedimentation at unit gravity. An increase in the modal sedimentation velocity and the percentage of rapidly sedimenting BFU-e was found in regenerating marrow as compared to normal marrow. Neither hypertransfusion-induced plethora nor administration of erythropoietin (Ep) during regeneration altered the changes from normal in the velocity sedimentation profile observed during regeneration. Separated marrow cells were pooled as rapidly sedimenting and slowly sedimenting and then examined for percentage of BFU-e in DNA synthesis and growth response in vitro to increasing concentrations of a partially purified Ep preparation. The percentage of BFU-e in DNA synthesis as determined by tritiated thymidine killing does not correspond to the BFU-e growth response to Ep in vitro . No difference in growth was noted between BFU-e from rapidly and slowly sedimenting normal marrow cells despite an increased percentage in DNA synthesis of normal BFU-e which sedimented rapidly. No significant difference in the percentage of BFU-e in DNA synthesis was found between the rapidly and slowly sedimenting subpopulations of regenerating BFU-e, but the latter had a reduced growth response to low concentrations of Ep.     

Production of granulocyte colony-stimulating factor and granulocyte/macrophage-colony-stimulating factor after interleukin-1 stimulation of marrow stromal cell cultures from normal or aplastic anemia donors.

We have studied stromal cell function in naive or interleukin-1 (IL-1)-stimulated (100 pg/ml) long-term marrow cultures (LTC) from 12 normal donors and 21 patients with severe aplastic anemia (AA). Conditioned media (CM) from normal LTC contained levels of erythroid burst-promoting activity (BPA) and granulocyte/macrophage (GM) colony-stimulating activity (CSA) comparable to those previously described (Migliaccio et al., [1990] Blood, 75:305-312). The addition of IL-1 to these cultures increased the level of CSA and, specifically, of granulocyte colony-stimulating factor (G-CSF) released. Anti-GM-CSF antibody neutralized BPA and CSA in normal naive LTC CM but only the CSA in the CM from IL-1-stimulated LTC. Since the concentrations of GM-CSF, as detected with a specific immunoassay, did not increase after IL-1 treatment, these data suggest that IL-1-stimulated cultures contain an unidentified growth factor having BPA. CM from AA stromal cells contained levels of CSA comparable to those observed in normal stromal cell CM but had significantly lower levels of BPA. Neither anti-GM-CSF nor anti-IL-3 antibodies neutralized the BPA in AA stromal cell CM. This activity may be related to that found in the CM of IL-1-treated normal stromal cells. In nearly 50% of stromal cell cultures of AA patients, addition of IL-1 failed to increase the BPA, CSA, or G-CSF. The presence of an inhibitor in naive or IL-1-treated AA stromal cell CM was excluded by adding the CM to IL-3-stimulated cultures. These findings suggest that G-CSF and GM-CSF genes are differentially regulated in the marrow microenvironment. Furthermore, a marrow microenvironment, deficient in BPA production and, in some cases, unresponsive to IL-1 could contribute to marrow failure in some patients with AA.     

Expression of tumor-associated surface membrane antigens on marrow CFU-s, CFC-gm, BFU-e, and brain CFU-s

Antiserum raised against a mouse mast cell line (FMP1) reacts with 90% to 100% of spleen colony-forming units (CFU-s), granulocyte/macrophage colony-forming cells (CFC-gm), erythroid burst-forming units (BFU-e), and 15% of nucleated marrow cells, using a complement-dependent cytotoxicity assay. We demonstrated that bone marrow, spleen, or thymus cells are able to absorb this activity from the antiserum. Although mouse brain cells have low reactivity with anti-FMP1 serum, the cytolysis level was reduced to background when antiserum was absorbed with brain cells. In addition, colony formation by marrow CFU-s, CFC-gm, and BFU-e was no longer prevented when the cells were incubated with brain-absorbed anti-FMP1 serum and complement. These findings suggest the presence of brain-associated antigens on CFU-s, CFC-gm, and BFU-e. To test whether a CFU-s accessory cell population in marrow is affected by treatment with anti-FMP1 serum and complement, antibody-treated marrow cells were mixed with large numbers of thymocytes and injected into recipient mice. Colony formation was not altered, indicating that the antiserum reacted directly with antigens on CFU-s and not on CFU-s accessory cells.     

Serum-free culture of primitive murine hemopoietic colony-forming cells

We report the effect of four sources of hemopoietic growth factors, alone or in combination, on colony growth in serum-free cultures of bone marrow from normal mice or marrow from mice pre-treated with 5-fluorouracil (5-FU-bm). The four supplements were: mouse spleen conditioned medium (SCM, a source of multi-lineage colony-stimulating activity, multi-CSA), human placental conditioned medium (HPCM, a source of synergistic activity), pregnant mouse uterus extract (PMUE, a source of M-CSA) and erythropoietin (Epo). First, in cultures of normal marrow, only PMUE and SCM induced significant colony growth when added alone. The majority of those colonies contained granulocytes and macrophages (myeloid colonies). In Epo-supplemented cultures, only SCM supported the growth of erythroid bursts and mixed erythroid-myeloid colonies. HPCM thus appears to be a poor source of multi-CSA. Second, in cultures of 5-FU-bm, few colonies developed if any of the above supplements were added alone. Only SCM + Epo together stimulated the formation of a low number of very large, mixed erythroid/myeloid/megakaryocyte colonies. HPCM, but not SCM, synergized with PMUE to augment myeloid colony numbers. Hence, SCM appears to be a poor source of synergistic activity (SA). In cultures of 5-FU-bm already supplemented with HPCM + PMUE, the addition of Epo did not change total colony numbers but did induce erythroid differentiation in one third of the colonies present. These data suggest that multi-CSA and SA may be expressed by different factors and that 5-FU pre-treated marrow contains: a population of primitive multipotential progenitors which form large, mixed colonies in the presence of SCM + Epo, and a larger Epo-sensitive population which also requires HPCM + PMUE to form mixed colonies.