Synergy between erythropoietin and stem cell factor during erythropoiesis can be quantitatively described without co-signaling effects

Synergistic interactions between cytokines underlie developmental processes fundamental to tissue and cellular engineering. However, a mechanistic understanding of the cell-specific and population-mediated effects is often lacking. In this study, we have investigated the synergistic generation of erythroid cells in response to erythropoietin (EPO) and stem cell factor (SCF). We have used a quantitative approach to determine if the effects of EPO and SCF superpose in a supra-additive fashion on the cell proliferation rate or on the death rate, suggesting a contribution from a joint cytokine effect (co-signaling). Primary mouse bone marrow hematopoietic cells and the stem cell-like FDCP-mix cell line were used to investigate the effects of EPO and SCF (individually or in combination) on erythroid output. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based cell-division tracking and mathematical modeling were used to measure cell type-specific proliferation and death rates. We observed a significant synergistic effect of EPO and SCF on the net generation of benzidine positive (erythroid) colony-forming cells, CD71++ (early erythroblasts) cells and TER-119+ (late erythroblasts and reticulocytes) cells in culture. When the observed increases in cell number were decomposed into proliferation and death rates, the cytokines were shown to act independently at different stages of erythroid development; SCF promoted the early proliferation of primitive cells, while EPO primarily promoted the survival of differentiating erythroid progenitor cells. Our analysis demonstrates that EPO and SCF have distinct and predominantly sequential effects on erythroid differentiation. This study emphasizes the necessity to separate proliferation rates from death rates to understand apparent cytokine synergies.     

Distinct functions of erythropoietin and stem cell factor are linked to activation of mTOR kinase signaling pathway in human erythroid progenitors

Erythropoietin (EPO) and Stem Cell Factor (SCF) have partially distinct functions in erythroid cell development. The primary functions of EPO are to prevent apoptosis and promote differentiation, with a minor role as a mitogen. On the other hand SCF acts primarily as a mitogenic factor promoting erythroid cell proliferation with a minor role in inhibition of apoptosis. The concerted effects of these two growth factors are responsible for guiding initial commitment, expansion and differentiation of progenitors. The aim of the study was to identify signaling elements pertinent to translational control and elucidate whether both cytokines can contribute to protein translation providing some functional redundancy as seen with respect to apoptosis. The current study focused on non-apoptotic functions of SCF mediated through mTOR/p70S6 leading to protein translation and cell proliferation. We utilized a human primary erythroid progenitors and erythroblasts that are responsive to EPO and SCF to investigate the activation of mTOR/p70S6 kinases and their downstream effectors, the pathway primarily responsible for protein translation. We showed that mTOR, p70S6 kinases and their downstream signaling elements 4EBP1 and S6 ribosomal protein are all activated by SCF but not by EPO in primary erythroid progenitors. We also found that SCF is the sole contributor to activation of the protein translational machinery and activation of mTOR/p70S6 pathway is confined to the proliferative phase of erythroid differentiation program. Altogether these results demonstrate that unlike the survival function which is supported by both EPO and SCF protein translation essential for proliferation is governed by only SCF.     

Measurement of generation-dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture.     

An in silico erythropoiesis model rationalizing synergism between stem cell factor and erythropoietin

Stem cell factor (SCF) and erythropoietin (EPO) are two most recognized growth factors that play in concert to control in vitro erythropoiesis. However, exact mechanisms underlying the interplay of these growth factors in vitro remain unclear. We developed a mathematical model to study co-signaling effects of SCF and EPO utilizing the ERK1/2 and GATA-1 pathways (activated by SCF and EPO) that drive the proliferation and differentiation of erythroid progenitors. The model was simplified and formulated based on three key features: synergistic contribution of SCF and EPO on ERK1/2 activation, positive feedback effects on proliferation and differentiation, and cross-inhibition effects of activated ERK1/2 and GATA-1. The model characteristics were developed to correspond with biological observations made known thus far. Our simulation suggested that activated GATA-1 has a more dominant cross-inhibition effect and stronger positive feedback response on differentiation than the proliferation pathway, while SCF contributed more to the activation of ERK1/2 than EPO. A sensitivity analysis performed to gauge the dynamics of the system was able to identify the most sensitive model parameters and illustrated a contribution of transient activity in EPO ligand to growth factor synergism. Based on theoretical arguments, we have successfully developed a model that can simulate growth factor synergism observed in vitro for erythropoiesis. This hypothesized model can be applied to further computational studies in biological systems where synergistic effects of two ligands are seen.     

Induction of foetal haemoglobin synthesis in erythroid progenitor stem cells: mediated by water‐soluble components of Terminalia catappa

Current novel therapeutic agents for the treatment of sickle cell anaemia (SCA) focus on increasing foetal haemoglobin (HbF) levels in SCA patients. Unfortunately, the only approved HbF‐inducing agent, hydroxyurea, has long‐term unpredictable side effects. Studies have shown the potential of plant compounds to modulate HbF synthesis in primary erythroid progenitor stem cells. We isolated a novel HbF‐inducing Terminalia catappa distilled water active fraction (TCDWF) from Terminalia catappa leaves that induced the commitment of erythroid progenitor stem cells to the erythroid lineage and relatively higher HbF synthesis of 9.2‐ and 6.8‐fold increases in both erythropoietin (EPO)‐independent and EPO‐dependent progenitor stem cells respectively. TCDWF was differentially cytotoxic to EPO‐dependent and EPO‐independent erythroid progenitor stem cell cultures as revealed by lactate dehydrogenase release from the cells. TCDWF demonstrated a protective effect on EPO‐dependent and not EPO‐independent progenitor cells. TCDWF induced a modest increase in caspase 3 activity in EPO‐independent erythroid progenitor stem cell cultures compared with a significantly higher (P?0.05) caspase 3 activity in EPO‐dependent ones. The results demonstrate that TCDWF may hold promising HbF‐inducing compounds, which work synergistically, and suggest a dual modulatory effect on erythropoiesis inherent in this active fraction. Copyright © 2014 John Wiley & Sons, Ltd.     

c-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

The expression of c-myc was analyzed in murine and human erythroblasts throughout their differentiation in vitro into reticulocytes. The murine cells were splenic erythroblasts from animals infected with the anemia strain of Friend virus (FVA cells). In FVA cells cultured without EPO, the c-myc mRNA and protein levels decrease sharply within 3 to 4 h, showing that continual EPO stimulation is required to maintain c-myc expression. When cultured with EPO, the c-myc mRNA level of FVA cells is raised within 30 min of exposure. The c-myc mRNA and protein reach maxima at 1 to 3 h, then decline slowly to very low levels by 18 h. In contrast, c-fos and c-jun mRNA levels are not regulated by EPO in FVA cells. The human cells analyzed were colony-forming units-erythroid, CFU-E, derived in vitro by the culture of peripheral blood burst-forming units-erythroid (BFU-E). When grown in EPO and insulin-like growth factor 1 (IGF-1) these cells differentiate into reticulocytes over 6 days rather than the 2 days required for murine cells, but the c-myc mRNA kinetics and response to EPO parallel those of mouse cells at similar stages of differentiation. Both IGF-1 and c-kit ligand (SCF) cause an additive increase in c-myc mRNA in human CFU-E in conjunction with EPO. These additive effects suggest that EPO, IGF-1, and SCF affect c-myc mRNA accumulation by distinct mechanisms. Addition of an antisense oligonucleotide to c-myc in cultures of human CFU-E specifically inhibited cell proliferation but did not affect erythroid cell differentiation or apoptosis. When human cells were grown in high SCF concentrations, an environment which enhances proliferation and retards differentiation, antisense oligonucleotide to c-myc strongly inhibited proliferation, but such inhibition did not induce differentiation. This latter result indicates that differentiation requires signals other than depression of c-myc and resultant depression of proliferation. © 1996 Wiley-Liss, Inc.     

The Notch2–Jagged1 interaction mediates stem cell factor signaling in erythropoiesis

Stem cell factor (SCF), the ligand for the c-kit receptor, is essential for the production of red blood cells during development and stress erythropoiesis. SCF promotes erythroblast proliferation and survival, while delaying erythroid differentiation through mechanisms that are largely unknown. In cultures of primary human differentiating erythroblasts, we found that SCF induces an increase in the expression of Notch2, a member of the Notch family implicated in the control of cell growth and differentiation. Functional inhibition of either Notch or its ligand Jagged1 inhibited the effects of SCF on erythroid cell expansion. SCF also induced the expression of Hes-1 and GATA-2, which may contribute to transduce Notch2 signals in response to SCF. Transduction of primary erythroid precursors with a dominant-negative Notch2 mutant inhibited both basal and SCF-mediated erythroblast expansion, and counteracted the effects of SCF on erythroblast differentiation. These findings provide a clue to understand the effects of increased proliferation and delayed differentiation elicited by SCF on the erythroid compartment and indicate Notch2 as a new player in the regulation of red cell differentiation.     

MLL-ENL cooperates with SCF to transform primary avian multipotent cells

The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic progenitors, we introduced the MLL-AF9 and MLL-ENL fusion proteins into primary chicken bone marrow cells. Both fusion proteins caused the sustained outgrowth of immature haematopoietic cells, which was strictly dependent on stem cell factor (SCF). The renewing cells have a long in vitro lifespan exceeding the Hayflick limit of avian cells. Analysis of clonal cultures identified the renewing cells as immature, multipotent progenitors, expressing erythroid, myeloid, lymphoid and stem cell surface markers. Employing a two-step commitment/differentiation protocol involving the controlled withdrawal of SCF, the MLL-ENL-transformed progenitors could be induced to terminal erythroid or myeloid differentiation. Finally, in cooperation with the weakly leukaemogenic receptor tyrosine kinase v-Sea, the MLL-ENL fusion protein gave rise to multilineage leukaemia in chicks, suggesting that other activated, receptor tyrosine kinases can substitute for ligand-activated c-Kit in vivo.     

Chaperonin 10 as an endothelial‐derived differentiation factor: Role of glycogen synthase kinase‐3

The anti‐inflammatory peptide early pregnancy factor/chaperonin 10 (cpn10) was identified by 2D‐electrophoresis/mass spectrometry as one of the proteins increased in human umbilical cord endothelial cells (HUVEC) after treatment with erythropoietin (EPO). EPO increased the amount of cpn10 released into the medium of HUVEC cultures, despite the absence of a secretory signal peptide. Although immunosupressive agents would represent an indirect advantage for red cell formation under conditions of infection and inflammation, it is possible that cpn10 may have direct effects on erythroid cells. We show that the chaperonin decreased cell proliferation in cultures of the erythroleukemia cell line K562 and increased the amounts of the erythroid differentiation markers glycophorin A and hemoglobin in TF‐1 cells. Nevertheless, cpn10 is not a specific erythroid cell differentiation factor, because monolayers of skin fibroblasts overexpressing cpn10 had significantly higher levels of the differentiation marker collagen I than normal fibroblasts. Nothing is known about the mechanism of action of cpn10 in its capacity as a general differentiation factor. An analysis of early changes taking place in K562 cells after incubation with cpn10 using antibody microarrays identified several phosphorylation events, including a decrease of GSK‐3α phosphorylation. Further studies in TF‐1 cells indicated that cpn10 decreased the phosphorylation of cofilin‐1 while stimulating that of GSK‐3β. Furthermore, glycophorin A production decreased in the presence of a GSK‐3 inhibitor in the same cells. These experiments support the idea that GSK‐3‐regulated signal transduction pathways are not only important for stem cell maintenance but may be involved in events controlling cell differentiation. J. Cell. Physiol. 219: 470–476, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of cytokine interactions by flow cytometry and factorial analysis

BACKGROUND: Multiple cytokines are required for the growth and development of hematopoietic cells. The effect of many cytokines depends on the activity of other signaling pathways. These interactions are quantified using factorial experimental design and analysis. METHODS: Human umbilical cord blood (HUCB) CD34+ cells were cultured in fully defined media containing various combinations of recombinant cytokines as defined by resolution IV factorial (2(7-3)(IV)) or full factorial (2(4)) design experiments. The cytokines studied were stem cell factor (SCF), interleukin (IL)-3, megakaryocyte growth and development factor (MGDF), granulocyte-colony stimulating factor (G-CSF), Flt-3 ligand, IL-6, IL-11, and erythropoietin (EPO). In vitro cell divisions were tracked by staining CD34+ cells with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, followed by flow cytometric analysis at 4 days of culture. In separate experiments, lineage commitment and differentiation were determined at 7 days by immunophenotype. RESULTS: In addition to the main effects of single cytokines, cytokine interactions were identified. There was a negative interaction between IL-3 and MGDF that resulted in a less than additive effect of these factors on erythroid and megakaryocytic development. The effect of Flt-3 ligand and SCF factor on CD34+ cell production was also less than additive, although the response to both cytokines was greater than single cytokines. The only positive interaction that was identified was between EPO and SCF, which resulted in the synergistic production of erythroid cells. CONCLUSIONS: Factorial analysis provides a powerful methodology to study the integration of multiple signals at the cellular and molecular level.     

Regulation of programmed death in erythroid progenitor cells by erythropoietin: effects of calcium and of protein and RNA syntheses.

Erythropoietin (EPO) retards DNA breakdown characteristic of programmed cell death (apoptosis) and promotes survival in erythroid progenitor cells. The mechanism by which EPO inhibits programmed death is unknown. In the well-characterized model of glucocorticoid-treated thymocytes, activation of a Ca2+/Mg(2+)-sensitive endonuclease and new protein and RNA syntheses have been found necessary for apoptosis. We examined the effects of EPO on the free intracellular calcium ion concentration ([Ca2+]i), and the roles of Ca2+ and RNA and protein syntheses on DNA cleavage in erythroid progenitor cells. The murine model of erythroid differentiation using Friend leukemia virus-infected proerythroblasts (FVA cells) was used. EPO did not affect the [Ca2+]i in FVA cells. Decreasing [Ca2+]i by extracellular Ca2+ chelation with EGTA facilitated DNA breakdown. Increasing [Ca2+]i with the calcium ionophore 4-bromo-A23187 increased DNA cleavage; however, DNA fragments generated by high [Ca2+]i were much larger than those seen in the absence of EPO or presence of EGTA. Increased [Ca2+]i also inhibited DNA breakdown to small oligonucleosomal fragments characteristic of cells cultured without EPO. However, no concentration of ionophore protected the high molecular weight DNA as did EPO. Cycloheximide inhibited DNA breakdown in a dose dependent manner in cultures lacking EPO, but two other protein synthesis inhibitors, pactamycin and puromycin, did not prevent DNA breakdown. Inhibition of RNA synthesis with actinomycin D did not prevent DNA breakdown. Cells with morphological characteristics similar to those reported in other cells undergoing programmed death accumulated in EPO-derived cultures. These studies demonstrate that although DNA cleavage and morphological changes are common to apoptotic cells, the roles for Ca2+ and protein and RNA syntheses are not universal and suggest that apoptosis can be regulated by different biochemical mechanisms in different cell types.     

Role of Caspase-10-P13tBID axis in erythropoiesis regulation

Red blood cell production is negatively controlled by the rate of apoptosis at the stage of CFU-E/pro-erythroblast differentiation, depending on the balance between erythropoietin (EPO) levels and activation of the Fas/FasL pathway. At this stage, activation of transient caspases through depolarization via mitochondrial outer membrane permeabilization (MOMP) is also required for terminal erythroid differentiation. Molecular mechanisms regulating the differential levels of MOMP during differentiation and apoptosis, however, remain poorly understood. Here we show a novel and essential role for the caspase-10-P13-tBID axis in erythroid terminal differentiation. Caspase-10 (but not caspase-8, which is activated during apoptosis) is activated at the early stages of erythroid terminal differentiation leading to the cleavage of P22-BID into P18-tBID, and later into P13-tBID. Erythropoietin (EPO) by inducing casein kinase I alpha (CKIα) expression, which in turn phosphorylates P18-tBID, prevents the generation of MYR-P15-tBID (leading to apoptosis) and allows the generation of P13-tBID by caspase-10. Unlike P15-tBID, P13-tBID is not myristoylated and as such, does not irreversibly anchor the mitochondrial membrane resulting in a transient MOMP. Likewise, transduction of a P13-tBID fragment induces rapid and strong erythroid terminal differentiation. Thus, EPO modulates the pattern of BID cleavage to control the level of MOMP and determines the fate of erythroblasts between apoptosis and differentiation. This pathway is impaired in 5q- myelodysplastic syndromes because of CK1α haplo-insufficiency and may contribute to erythroid differentiation arrest and high sensitivity of this disease to lenalidomide (LEN).Subject terms: Cell biology, Proteolysis

Model describing the critical impact of EPO levels on P18-tBID phosphorylation through up-regulation of CK1α, which determines the BID cleavage pattern (differentiating P13-tBID or apoptotic MYR-P15-tBID) and thus the immature erythroblasts’ fate.     

Identification of key genes responsible for cytokine-induced erythroid and myeloid differentiation and switching of hematopoietic stem cells by RAGE

We utilized a unique culture system to analyze the expression patterns of gene, protein, and cell surface antigen, and the biological process of the related genes in erythroid and myeloid differentiation and switching of hematopoietic stem cells （HSCs） in response to cytokine alterations. Gene-specific fragments （266） identified from five populations of cytokine-stimulated HSCs were categorized into three groups： （1） expressed specifically in a single cell population; （2） expressed in two cell populations, and （3） expressed in three or more populations. Of 145 defined cDNAs, three （2%） were novel genes. Protein two-dimensional gel electrophoresis and flow cytometry analyses showed overlapped and distinguished protein expression profiles in the cell populations studied. Biological process mapping of mRNAs expressed in erythroid and myeloid lineages indicated that mRNAs shared by both lineages attended ＇core processes,＇ whereas genes specifically expressed in either lineage alone were related to specific processes or cellular maturation. Data from this study support the hypothesis that committed HSCs （El4 or G14） cells can still be redirected to develop into myeloid or erythroid cells when erythropoietin （EPO） is replaced with granulocyte-colony stimulating factor （G-CSF） under erythroid-cultured condition or G-CSF with EPO in myeloid-cultured environment, respectively. Our results suggest that genes or proteins co-expressed in erythroid and myeloid lineages may be essential for the lineage maintenance and switching in hematopoiesis.     

Apoptosis of human erythroid colony-forming cells is decreased by stem cell factor and insulin-like growth factor I as well as erythropoietin

To clarify the manner by which erythropoietin (EP), stem cell factor (SCF), or insulin-like growth factor I (IGF-I) regulate erythropoiesis, apoptosis of human erythroid progenitor cells was investigated. Human burst-forming units-erythroid (BFU-E) partially purified from peripheral blood were cultured for 6 days to generate erythroid colony-forming cells (ECFC), which consist mainly of colony-forming units-erythroid (CFU-E). The cells were labeled with [₃H]thymidine, incubated in serum-free liquid media, at 37°C, for 16 h, and the pattern of DNA breakdown was analyzed by agarose gel electrophoresis. When these cells were incubated without EP, 70% of the total cellular DNA was broken down into DNA fragments of less than 5 kilobases and nuclear condensation and fragmentation, characteristic of apoptosis, were evident. While EP greatly reduced the amount of DNA breakdown to 23%, SCF and IGF-I each reduced the amount of DNA breakdown to 38–46% and, when added together, to 24%. Dose-response experiments with SCF and IGF-I showed a dose-dependent reduction in DNA fragmentation at concentrations that stimulate colony formation in serum-free semisolid cultures. Finally, assays of ECFC performed by the plasma clot method, after serum-free liquid culture, at 37°C, for 16 h, demonstrated marked protection of erythroid colony-forming capacity by SCF or IGF-I in the absence of EP, as well as by EP itself. These data indicate that human erythroid progenitor cells undergo apoptosis which is reduced by SCF and IGF-I as well as EP and suggest that the control of apoptosis by each of these factors has a prominent role in the regulation of erythropoiesis. © 1993 Wiley-Liss, Inc.     

脐带血造血干/祖细胞体外分化为不同阶段红系祖细胞的动力学观察

目的:探讨体外培养脐带血单个核细胞定向诱导分化为不同阶段红系祖细胞的动力学变化情况。方法:用0.5%甲基纤维素沉降脐带血红细胞及人淋巴细胞分离液密度梯度离心法得到单个核细胞,在含EPO、SCF、IGF-1等细胞因子的无血清培养体系中诱导其定向分化为红系祖细胞,观察细胞增殖、存活率、细胞集落形成情况,并检测不同阶段细胞红系特异性表面标志CD71和CD235a的表达。结果:随着培养时间的延长,细胞数逐渐增多,14 d细胞可扩增140倍左右,收集诱导后的细胞进行瑞氏吉姆萨染色,可见大量红系祖细胞,诱导后的细胞集落形成能力强,形成的克隆大部分为红系集落。诱导过程中,14 d前CD71、CD235a的表达逐渐增高。按细胞表面标志表达的不同可将诱导的细胞分为4群,分别对应红系祖细胞的不同阶段;随着诱导天数的增加,各时间点细胞对应的早期红系祖细胞群(P2、P3)比例逐渐下降,中晚期红系祖细胞群(P4、P5)的比例逐渐上升。结论:无血清培养基添加细胞因子组合的红系诱导培养体系可较好地诱导扩增红系祖细胞,流式分选可获得相对均一而处于不同分化阶段的红系祖细胞群体。获得了红系祖细胞体外分化的动力学数据,为今后进一步优化红系诱导分化体系获得均一的红系祖细胞奠定了基础,并对未来利用干细胞制备均一的红系祖细胞应用于临床治疗有一定的指导作用。     

Stem cell factor and granulocyte colony-stimulating factor promote neuronal lineage commitment of neural stem cells

Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) were originally discovered as growth factors for hematopoietic stem cells (HSCs). It has been well defined that SCF and G-CSF contribute to regulation of lineage commitment for HSCs. However, little is known about whether SCF and G-CSF play roles in the determination and differentiation of neural stem cells (NSCs). Here we demonstrate the novel function of SCF and G-CSF in controlling cell cycle and cell fate determination of NSCs. We also observe that SCF and G-CSF promote neuronal differentiation and inhibit astroglial differentiation at the early stage of differentiation. In addition, our research data reveal that SCF in combination with G-CSF has a dual function in promoting cell cycle exit and directing neuronal fate commitment at the stage of NSC dividing. This coordination effect of SCF+G-CSF on cell cycle arrest and neuronal differentiation is through enhancing neurogenin 1 (Ngn1) activity. These findings extend current knowledge regarding the role of SCF and G-CSF in the regulation of neurogenesis and provide insights into the contribution of hematopoietic growth factors to brain development and remodeling.     

Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation.

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.