Colony-forming Unit-erythroid Colonies Research Articles

Nitric Oxide Synthase Dependency in Hydroxyurea Inhibition of Erythroid Progenitor Growth.

Hydroxyurea (HU) causes nitric oxide (NO) bioactivation, acting as both a NO donor and a stimulator of NO synthase (NOS). To examine whether HU effects are NO mediated by chemical degradation or enzymatic induction, we studied human and mouse erythroid cells during proliferation, apoptosis, and differentiation. The HU and NO donor demonstrated persisted versus temporary inhibition of erythroid cell growth during differentiation, as observed by γ- and β-globin gene expression. HU decreased the percentage of erythroleukemic K562 cells in the G2/M phase that was reversed by N-nitro l-arginine methyl ester hydrochloride (L-NAME). Besides activation of endothelial NOS, HU significantly increased apoptosis of K562 cells, again demonstrating NOS dependence. Administration of HU to mice significantly inhibited colony-forming unit-erythroid (CFU-E), mediated by NOS. Moreover, burst-forming-units-erythroid (BFU-E) and CFU-E ex vivo growth was inhibited by the administration of nitrate or nitrite to mice. Chronic in vivo NOS inhibition with L-NAME protected the bone marrow cellularity despite HU treatment of mice. NO metabolites and HU reduced the frequency of NOS-positive cells from CFU-E and BFU-E colonies that was reverted by NOS inhibition. HU regulation of the G2/M phase, apoptosis, differentiation, cellularity, and NOS immunoreactive cells was NOS dependent. Inhalation of NO therapy as well as strategies to increase endogenous NO production could replace or enhance HU activity.

TET2 Deficiency Led to Expansion of Dysfunctional Erythroid Progenitors

Erythrocyte development is a complex process involving multiple developmental states that include erythroid progenitors, burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells and erythroid precursors, proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts in the bone marrow. Orthochromatic erythroblasts expel nucleus to become reticulocytes which mature into erythrocytes in blood stream. Defects at any of these developmental stages will lead to anemia. Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by cytopenia and ineffective hematopoiesis. Anemia is the defining cytopenia in the majority of patients with MDS, yet the molecular mechanisms for dyserythropoiesis in MDS remain largely unclear. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is the most common mutation in MDS.In the present study, we explored shRNA-mediated knockdown approach in human CD34+ cell to study the role of TET2 in human erythropoiesis. We show that TET2 is downregulated starting at the CFU-E stage. Growth curves of purified CFU-E cells show no difference between control and TET2 knockdown cells until day 6. An increased proliferation of TET2 knockdown-CFU-E cells is noted starting at day 7. Interestingly, while the growth of control CFU-E cells plateaued on day 11 with completion of terminal erythroid differentiation and enucleation, TET2 knockdown CFU-E cells continued their growth for additional 13 days. Flow cytometry and cytospin morphologic analyses revealed that by day 11 all control cells differentiated into GPA+ late stage erythroblasts or reticulocytes. In marked contrast, about 30 % of TET2 knockdown cells are still GPA- but CD71+, IL-3R-, CD34-, CD36+, phenotypically resembling erythroid progenitor CFU-E cells. Surprisingly, in contrast to freshly purified control and TET2 knockdown CFU-E cells that generate colonies in methylcellulose colony forming assays, these cells fail to give rise to CFU-E colonies. We thus termed these late proliferating cells as “TET2 knockdown marker CFU-E”. Further characterization of the “TET2 knockdown marker CFU-E” cells revealed that in contrast to control CFU-E, the “TET2 knockdown marker CFU-E” exhibit following distinct features: 1) they had lower proliferative potential; 2) their proliferation required both EPO and SCF (while control CFU-E cells only require EPO); 3) they underwent extensive apoptosis in the absence of SCF; 4) they failed to undergo terminal erythroid differentiation. These findings demonstrate that TET2 knockdown led to expansion of dysfunctional erythroid progenitors.To explore the underlying molecular mechanisms, we performed RNA-seq analysis on control CFU-E, TET2 knockdown CFU-E and “TET2 knockdown marker CFU-E” cells. Bioinformatics analysis revealed that while only about 50 genes were differentially expressed between control CFU-E and TET2 knockdown CFU-E, about 400 genes were differentially expressed between control CFU-E and “TET2 knockdown marker CFU-E”. Gene set enrichment analysis revealed upregulation of pathways associated with apoptosis/cell death. Notably, FAS (CD95), the cell surface death receptor previously shown to be upregulated in MDS bone marrow cells, was among the top five upregulated apoptosis associated genes. The increased surface expression of FAS was also noted in TET2-knockdown cells as assessed by flow cytometry. Furthermore, the increased surface expression of FAS was detected in primary bone marrow erythroblasts of MDS patients with TET2 mutation. Our findings uncovered previously unknown roles of TET2 and its potential underlying molecular mechanisms. As the phenotypic changes upon TET2 knockdown resemble the characteristic pathological changes of MDS, our findings suggest the involvement of TET2 deficiency in the pathophysiology of MDS. DisclosuresAli:Onconova Therapeutics: Consultancy; Kura Oncology: Consultancy. Raza:Celgene Inc.: Research Funding; Syros Pharmaceuticals: Research Funding; Onconova Therapeutics: Research Funding, Speakers Bureau; Genoptix: Speakers Bureau; Kura Oncology: Research Funding; Novartis: Speakers Bureau; Janssen R&D: Research Funding.

Clostridium perfringens \u03b1-toxin impairs erythropoiesis by inhibition of erythroid differentiation

Clostridium perfringens α-toxin induces hemolysis of erythrocytes from various species, but it has not been elucidated whether the toxin affects erythropoiesis. In this study, we treated bone marrow cells (BMCs) from mice with purified α-toxin and found that TER119+ erythroblasts were greatly decreased by the treatment. A variant α-toxin defective in enzymatic activities, phospholipase C and sphingomyelinase, had no effect on the population of erythroblasts, demonstrating that the decrease in erythroblasts was dependent of its enzymatic activities. α-Toxin reduced the CD71+TER119+ and CD71–TER119+ cell populations but not the CD71+TER119− cell population. In addition, α-toxin decreased the number of colony-forming unit erythroid colonies but not burst-forming unit erythroid colonies, indicating that α-toxin preferentially reduced mature erythroid cells compared with immature cells. α-Toxin slightly increased annexinV+ cells in TER119+ cells. Additionally, simultaneous treatment of BMCs with α-toxin and erythropoietin greatly attenuated the reduction of TER119+ erythroblasts by α-toxin. Furthermore, hemin-induced differentiation of human K562 erythroleukemia cells was impaired by α-toxin, whereas the treatment exhibited no apparent cytotoxicity. These results suggested that α-toxin mainly inhibited erythroid differentiation. Together, our results provide new insights into the biological activities of α-toxin, which might be important to understand the pathogenesis of C. perfringens infection.

Bone marrow hematopoietic stem cells behavior with or without growth factors in trauma hemorrhagic shock.

Background:Hemorrhagic shock (HS) is the major leading cause of death after trauma. Up to 50% of early deaths are due to massive hemorrhage. Excessive release of pro-inflammatory cytokine and hypercatecholamine induces hematopoietic progenitor cells (HPCs) apoptosis, leading to multiorgan failure and death. However, still, result remains elusive for hematopoietic stem cells (HSCs) behavior in trauma HS (T/HS).Objectives:Therefore, our aim was to evaluate the in vitro HSCs behavior with or without recombinant human erythropoietin (rhEPO), recombinant human granulocyte macrophage-colony-stimulating factor (rhGM-CSF), recombinant human interleukin-3 (rhIL-3) alone, and combination with rhEPO + rhGM-CSF + rhIL-3 (EG3) in T/HS patients.Methodology:Bone marrow (BM) aspirates (n = 14) were collected from T/HS patients, those survived on day 3. BM cells were cultured for HPCs: Colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, monocyte/macrophage colonies growth. HPCs were counted with or without rhEPO, rhGM-CSF, rhIL-3 alone, and combination with EG3 in T/HS patients.Results:BM HSCs growth significantly suppressed in T/HS when compared with control group (P < 0.05). In addition, CFU-E and BFU-E colony growth were increased with additional growth factor (AGF) (rhEPO, rhGM-CSF, and rhIL-3) as compared to baseline (without AGF) (P < 0.05).Conclusion:Suppressed HPCs may be reactivated by addition of erythropoietin, GM-CSF, IL-3 alone and with combination in T/HS.

Mesenchymal stem cells reverse trauma and hemorrhagic shock-induced bone marrow dysfunction

BackgroundLung contusion (LC) followed by hemorrhagic shock (HS) causes persistent bone marrow (BM) dysfunction lasting up to 7 d after injury. Mesenchymal stem cells (MSCs) are multipotent cells that can hasten healing and exert protective immunomodulatory effects. We hypothesize that MSCs can attenuate BM dysfunction after combined LCHS. Materials and methodsMale Sprague–Dawley rats (n = 5–6 per group) underwent LC plus 45 min of HS (mean arterial pressure of 30–35). Allogeneic MSCs (5 × 106 cells) were injected intravenously after resuscitation. At 7 d, BM was analyzed for cellularity and growth of hematopoietic progenitor cell (HPC) colonies (colony-forming unit–erythroid; burst-forming unit–erythroid; and colony-forming unit–granulocyte, erythrocyte, monocyte, megakaryocyte). Flow cytometry measured %HPCs in peripheral blood; plasma granulocyte colony-stimulating factor (G-CSF) levels were measured via enzyme-linked immunosorbent assay. Data were analyzed by one-way analysis of variance followed by the Tukey multiple comparison test. ResultsAs previously shown, at 7 d, LCHS resulted in 22%, 30%, and 24% decreases in colony-forming unit–granulocyte, erythrocyte, monocyte, megakaryocyte, burst-forming unit–erythroid, and colony-forming unit–erythroid colony growth, respectively, versus naive. Treatment with MSCs returned all BM parameters to naive levels. There was no difference in %HPCs in peripheral blood between groups; however, G-CSF remained increased up to 7 d after LCHS. MSCs returned G-CSF to naive levels. Plasma from animals receiving MSCs was not suppressive to the BM. ConclusionsOne week after injury, the persistent BM dysfunction observed in animals undergoing LCHS is reversed by treatment with MSCs with an associated return of plasma G-CSF levels to normal. Plasma from animals undergoing LCHS plus MSCs was not suppressive to BM cells in vitro. Treatment with MSCs after injury and shock reverses BM suppression and returns plasma G-CSF levels to normal.

Isolation and transcriptome analyses of human erythroid progenitors: BFU-E and CFU-E

AbstractBurst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45+GPA−IL-3R−CD34+CD36−CD71low and CD45+GPA−IL-3R−CD34−CD36+CD71high phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity of ∼90%. The BFU-E colony forming ability of CD45+GPA−IL-3R−CD34+CD36−CD71low cells required stem cell factor and erythropoietin, while the CFU-E colony forming ability of CD45+GPA−IL-3R−CD34−CD36+CD71high cells required only erythropoietin. Bioinformatic analysis of the RNA-sequencing data revealed unique transcriptomes at each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow, cord blood, and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematologic disease. Our data provides an important resource for future studies of human erythropoiesis.

The Erythropoietin Receptor Regulates The Number Of Cell Divisions and The Duration Of Erythroblast Terminal Differentiation By Regulating Erythroblast Iron

Signaling by the erythropoietin receptor (EpoR) is essential for the survival of definitive colony-forming unit-erythroid (CFU-e) progenitors and their erythroblast progeny. Here we used EpoR-null embryos to ask whether EpoR signaling might also exert essential non-survival functions in erythropoiesis.To address this, we rescued EpoR-null fetal liver cells from death by transducing them in vitro with either the anti-apoptotic protein Bcl-xL, or, as control, with the wild-type EpoR. The Bcl-xL-transduced EpoR-null cells survived, expressed hemoglobin and underwent morphological erythroid maturation and enucleation. However, unlike exogenous EpoR, exogenous Bcl-xL was unable to support the formation of EpoR-null CFU-e colonies in methylcellulose. The absence of colonies was explained by the finding that the Bcl-xL-transduced progenitors underwent fewer cell divisions than equivalent EpoR-transduced cells (1.1 vs. 2.9 in 24 hr, respectively) and had a slower rate of intra-S phase DNA synthesis, suggesting longer S phase duration. Multispectral imaging showed that the Bcl-xL-transduced cells matured prematurely, attaining smaller cell and nuclear size and a lower nuclear/cytoplasmic ratio at earlier time points than EpoR-transduced cells. Premature maturation was also evident by flow cytometric analysis. Thus, EpoR-null fetal liver cells in vivo arrest in their differentiation at the transition from subset S0 (Ter119-neg CD71-low) to S1 (Ter119-neg CD71-high) (Pop et al, PLoS Biology 2010). Rescue with EpoR in vitro allows EpoR-null progenitors to resume differentiation, sequentially upregulating CD71 and Ter119. By contrast, rescue of EpoR-null cells with Bcl-xL results in their premature upregulation of Ter119 and failure to upregulate CD71 to high levels. The cell cycle and differentiation deficits in Bcl-xL-supported, EpoR-null erythropoiesis were associated with a slower loss of DNA methylation from the erythroid genome, and with slower erythroid gene transcription.CD71 (the transferrin receptor) is a known target of EpoR and Stat5 signaling. We asked whether the deficits of EpoR-null erythropoiesis might be the result of low cell surface CD71 and the consequent reduced iron transport. In support of this hypothesis, we found that EpoR-null fetal liver cells that are transduced with both CD71 and Bcl-xL resume the normal maturation rate characteristic of EpoR-supported differentiation, as judged by multispectral imaging measurements of cell size and nuclear/cytoplasmic ratio. Further, we were able to restore rapid S phase to Bcl-xL-transduced EpoR-/- erythroblasts by culturing them in the presence of the cell-permeant iron chelator Fe-SIH (salicylaldehyde isonicotinoyl hydrazone), which is able to supply the cell interior with iron even in the absence of CD71 (Figure 1). We suggest that EpoR-mediated upregulation of CD71 at the onset of erythroid terminal differentiation determines the number and duration of erythroblast cell divisions by regulating iron homeostasis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Isolation and Functional Characterization of Human Erythroid Progenitors: BFU-E and CFU-E

Abstract 1028The two committed erythroid progenitor populations that have been functionally defined by colony assays are burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E). While significant progress has been made in defining these two progenitor populations in the murine system, their characterization in the human system is incomplete. To address this issue, we have characterized the dynamic changes in surface expression levels of number of proteins including CD34, c-kit, IL-3R, CD36, CD71, GPA and CD45 during proliferation of purified human CD34+ cells from cord blood during the first phase of the two-phase in vitro erythroid culture system. In the presence of stem cell factor, IL-3 and erythropoietin during this phase, CD34+ cells differentiate first into BFU-E and then into CFU-E during 7 days of culture with peak levels of BFU-E at day 4 and of CFU-E at day 6. During this period of time, the expression levels of CD34 and IL-3R decreased, while that that of CD36 and CD71 increased. CD45 was expressed during the entire 7 day culture period while there was no expression of GPA. Based on these findings, we sorted pure populations of CD34+CD36−IL3-R+ and CD34− CD36+IL-3R− cells and characterized their behavior in colony forming assays. The sorted CD34+CD36−IL3-R+ population gave rise to BFU-E colonies while CD34− CD36+IL-3R− population gave rise CFU-E colonies, both at a purity of over 80%. The identity of the sorted BFU-E and CFU-E cells was further supported by their differential responsiveness to dexamethasone and lenalidomide (Narla A et al Blood 2011), with increased proliferation BFU-E population by dexamethasone and increased proliferation of CFU-E by lenalidomide. These findings were further validated by isolation of pure populations of BFU-E and CFU-E from primary human bone marrow based on the identified markers. The ability to isolate pure populations of human BFU-E and CFU-E progenitors should enable detailed molecular and cellular characterization of these distinct erythroid progenitor populations. Furthermore, enumeration of the number of these progenitor populations in human bone marrow may help in delineating mechanisms of disordered erythropoiesis in various disorders such as bone marrow failure syndromes. Disclosures:No relevant conflicts of interest to declare.

Association Between Elevated Prolactin Levels and Circulating Erythroid Precursors in Dialyzed Patients

Abstract. The prolactin (PRL) receptor (R), a member of the cytokine hemopoietin receptor superfamily, has been shown to activate early differentiation steps along the erythroid pathway. In particular PRL, a product of bone marrow stroma, induces functional erythropoietin (EPO)-R on CD34+ hemopoietic progenitors. In this study, expression of EPO-R mRNA and responsiveness to EPO were assessed on enriched hemopoietic progenitor cells (HPC) from seven hyperprolactinemic and three normoprolactinemic patients and two normal subjects. Expression of EPO-R mRNA by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was found in HPC of four out of seven hyperprolactinemic patients but not in normoprolactinemic patients or normal donors. Development of EPO-dependent Colony Forming Unit-Erythroid (CFU-E) colonies in semi-solid medium was observed only in hyperprolactinemic patients (six out of seven). A much higher number of CFU-E colonies was observed in the four patients with a positive EPO-R message. We conclude from these data that abnormally high levels of PRL may increase the number of EPO-responsive hemopoietic precursors in vivo as they do in vitro. Since hyperprolactinemia associates in these patients with depressed EPO production, it may be regarded as a compensatory mechanism for the reduced availability of the hemopoietic factor.

Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development

To find a human cell line that could support differentiation of human embryonic stem cells (hESCs) into hematopoietic cells. To determine in detail the expression profiles of the beta-like globin genes in hESC-derived erythroid cells. FH-B-hTERT, a human fetal liver-derived cell line, and S17, a mouse bone marrow stromal cell line, were used as stromas to induce the differentiation of hESC into hematopoietic cells. The number of hematopoietic progenitors and surface antigen expression were monitored during time-course experiments using colony assays and flow cytometry. Globin expression patterns in individual erythroid colonies were determined by real-time quantitative reverse transcriptase polymerase chain reaction. Comparison of coculture of hESCs with FH-B-hTERT or S17 cells revealed that the fraction of CD34(+) cells and the number of clonogenic progenitors per 250,000 cells plated were higher with FH-B-hTERT than with S17. Analysis of beta-like globin expression in individual burst-forming unit erythroid and colony-forming unit erythroid colonies revealed that erythroid cells derived from hESC cocultured for 8 to 21 days on either FH-B-hTERT or S17 produced epsilon- and gamma-globin mRNAs in similar amounts. With increasing time in coculture, the mean ratio of gamma/epsilon increased by more than 10-fold on both S17 and FH-B-hTERT stroma. Importantly, beta-globin expression was barely detectable at all time point examined. FH-B-hTERT can induce hESCs differentiation into hematopoietic cells more efficiently than S17. In vitro differentiation of hESCs recapitulates the epsilon-globin to gamma-globin switch but not the gamma-globin to beta-globin switch that occurs around birth. This experimental system will be useful for studying the regulation of globin gene expression during early human hematopoiesis.

Effect of l-carnitine on erythroid colony formation in mouse bone marrow cells

l-Carnitine can alleviate uraemic anaemia in haemodialysis patients by improving erythrocyte membrane functions or erythropoiesis, which are depressed under uraemic conditions. l-Carnitine and palmitoyl-l-carnitine were reported to increase the formation of colony-forming unit-erythroid (CFU-E) colonies in cultures of fetal mouse liver cells, an effect that depended on the concentration of palmitoyl-l-carnitine but not of l-carnitine. In this study, we investigated l-carnitine's effect on CFU-E colony formation in cell cultures of mouse bone marrow cells. Bone marrow from normal female mice was placed in 35 mm culture dishes containing a medium composed of methylcellulose and various nutrients. The dishes were incubated for 48 h, and the colonies of erythroblasts, which were differentiated from CFU-E, consisting of >/=8 cells, were counted in each dish using an inverted microscope. The numbers of CFU-E colonies correlated well with both the initial numbers of bone marrow cells and concentrations of recombinant human erythropoietin (rhEPO) in the methylcellulose medium. In the presence of 0.5 or 1.0 IU/ml of rhEPO, l-carnitine at concentrations of 200 and 400 micromol/l significantly enhanced CFU-E colony formation (P<0.001). l-Carnitine significantly increased the number of CFU-E colonies in mouse bone marrow cell cultures. This finding suggests that l-carnitine stimulates erythropoiesis, partially accounting for its mitigating effect on renal anaemia.

Essential Role of Synoviolin in Embryogenesis

We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno(-/-)) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno(-/-) fetal liver. Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno(-/-) embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno(-/-) mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno(+/+) mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.

MDYRK3 kinase is expressed selectively in late erythroid progenitor cells and attenuates colony-forming unit–erythroid development

AbstractDYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1, an inhibitor of cell cycle progression in yeast. At present, mDYRK-3 and mDYRK-2 have been cloned, and mDYRK-3 has been characterized with respect to kinase activity, expression among tissues and hematopoietic cells, and possible function during erythropoiesis. In sequence, mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a, but is 91.3% identical overall to hDYRK-3. Catalytically, mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro. Expression of mDYRK-1a, mDYRK-2, and mDYRK-3 was high in testes, but unlike mDYRK1a and mDYRK 2, mDYRK-3 was not expressed at appreciable levels in other tissues examined. Among hematopoietic cells, however, mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells. In developmentally synchronized erythroid progenitor cells, expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone), and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver. Interestingly, antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit–erythroid colony formation. Thus, it is proposed that mDYRK-3 kinase functions as a lineage-restricted, stage-specific suppressor of red cell development.

Association between elevated prolactin levels and circulating erythroid precursors in dialyzed patients.

The prolactin (PRL) receptor (R), a member of the cytokine hemopoietin receptor superfamily, has been shown to activate early differentiation steps along the erythroid pathway. In particular PRL, a product of bone marrow stroma, induces functional erythropoietin (EPO)-R on CD34+ hemopoietic progenitors. In this study, expression of EPO-R mRNA and responsiveness to EPO were assessed on enriched hemopoietic progenitor cells (HPC) from seven hyperprolactinemic and three normoprolactinemic patients and two normal subjects. Expression of EPO-R mRNA by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was found in HPC of four out of seven hyperprolactinemic patients but not in normoprolactinemic patients or normal donors. Development of EPO-dependent Colony Forming Unit-Erythroid (CFU-E) colonies in semi-solid medium was observed only in hyperprolactinemic patients (six out of seven). A much higher number of CFU-E colonies was observed in the four patients with a positive EPO-R message. We conclude from these data that abnormally high levels of PRL may increase the number of EPO-responsive hemopoietic precursors in vivo as they do in vitro. Since hyperprolactinemia associates in these patients with depressed EPO production, it may be regarded as a compensatory mechanism for the reduced availability of the hemopoietic factor.

An Agonist Murine Monoclonal Antibody to the Human c-Mpl Receptor Stimulates Megakaryocytopoiesis

Thrombopoietin (TPO) is a hematopoietic growth factor that stimulates megakaryocytopoiesis and platelet production in vivo and promotes the development of identifiable megakaryocytes in vitro. We have developed a murine monoclonal antibody, BAH-1, raised against human megakaryocytic cells, which specifically recognizes the c-Mpl receptor and shows agonist activity by stimulating megakaryocytopoiesis in vitro. BAH-1 antibody specifically binds to platelets and to recombinant c-Mpl with high affinity. Similar to TPO, BAH-1 alone supported the formation of colony-forming unit-megakaryocyte (CFU-MK) colonies. The combination of BAH-1 plus interleukin-3 or of BAH-1 plus human TPO significantly increased the number of human CFU-MK colonies. In addition, BAH-1 monoclonal antibody stimulated the proliferation and maturation of primary bone marrow megakaryocytes in a dynamic heterogeneous liquid culture system. Individual large megakaryocytes as well as small megakaryocytic cells were observed in cultures of CD34+ CD41+cells in the presence of BAH-1 antibodies. Similar to TPO, BAH-1 antibody induced a significant response of murine immature megakaryocytes as observed by an increase in the detectable numbers of acetylcholinesterase-positive megakaryocytes. No effects of BAH-1 antibody were observed on colony-forming unit–granulocyte-macrophage, burst-forming unit-erythroid, or colony-forming unit-erythroid colonies. In vivo studies showed that BAH-1, alone or in combination with TPO, expands the numbers of megakaryocytic progenitor cells in myelosuppressed mice. This antibody should prove useful in understanding the structure-function aspects of the c-Mpl receptor as well as in evaluating the effects of the sustained activation of this receptor in preclinical models of severe thrombocytopenia. © 1998 by The American Society of Hematology.

An Agonist Murine Monoclonal Antibody to the Human c-Mpl Receptor Stimulates Megakaryocytopoiesis

Thrombopoietin (TPO) is a hematopoietic growth factor that stimulates megakaryocytopoiesis and platelet production in vivo and promotes the development of identifiable megakaryocytes in vitro. We have developed a murine monoclonal antibody, BAH-1, raised against human megakaryocytic cells, which specifically recognizes the c-Mpl receptor and shows agonist activity by stimulating megakaryocytopoiesis in vitro. BAH-1 antibody specifically binds to platelets and to recombinant c-Mpl with high affinity. Similar to TPO, BAH-1 alone supported the formation of colony-forming unit-megakaryocyte (CFU-MK) colonies. The combination of BAH-1 plus interleukin-3 or of BAH-1 plus human TPO significantly increased the number of human CFU-MK colonies. In addition, BAH-1 monoclonal antibody stimulated the proliferation and maturation of primary bone marrow megakaryocytes in a dynamic heterogeneous liquid culture system. Individual large megakaryocytes as well as small megakaryocytic cells were observed in cultures of CD34+ CD41+cells in the presence of BAH-1 antibodies. Similar to TPO, BAH-1 antibody induced a significant response of murine immature megakaryocytes as observed by an increase in the detectable numbers of acetylcholinesterase-positive megakaryocytes. No effects of BAH-1 antibody were observed on colony-forming unit–granulocyte-macrophage, burst-forming unit-erythroid, or colony-forming unit-erythroid colonies. In vivo studies showed that BAH-1, alone or in combination with TPO, expands the numbers of megakaryocytic progenitor cells in myelosuppressed mice. This antibody should prove useful in understanding the structure-function aspects of the c-Mpl receptor as well as in evaluating the effects of the sustained activation of this receptor in preclinical models of severe thrombocytopenia.© 1998 by The American Society of Hematology.

Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation.

Production of mature erythrocytes requires multiple growth factors, but we do not know how their actions are coordinated. Here we show that erythroid progenitors from erythropoietin receptor (Epo-R)-/- fetal livers, infected in vitro with a retrovirus expressing the wild-type Epo-R, require addition of both Epo and stem cell factor (SCF) to form colony-forming unit erythroid (CFU-E) colonies. Thus, a functional interaction between KIT and the Epo-R, similar to what we reported in cultured cells, is essential for the function of CFU-E progenitors. In contrast, CFU-E colony formation in vitro by normal fetal liver progenitors requires only Epo; the essential interaction between activated KIT and the Epo-R must have occurred in vivo before or at the CFU-E progenitor stage. Using truncated dominant-negative mutant Epo-Rs, we show that KIT does not activate the Epo-R by inducing its dimerization, but presumably does so by phosphorylating tyrosine residue(s) in its cytosolic domain. By expressing mutant Epo-Rs containing only one of eight cytosolic tyrosines, we show that either tyrosine residue Y464 or Y479 suffices for Epo-dependent cell proliferation. However, only Epo-R F7Y479 is capable of supporting erythroid colony formation when expressed in (Epo-R)-/- fetal liver cells, indicating that Y464 either cannot send a differentiation signal or fails to respond to SCF/KIT activation. This work employs a novel experimental system to study the function of growth factors and their receptors in normal hematopoiesis.

Effects of spaceflight on rat erythroid parameters.

Hematologic studies were performed on 21 ground control rats and 21 rats flown during the Spacelab Life Sciences-2 14-day mission. Group A (n = 5) was used to collect blood in flight and 9 days postflight, group B (n = 5) was injected with recombinant human erythropoietin (rhEpo), group C (n = 5) received saline as a control, and group D (n = 6) was killed in flight and tissues were collected. Results indicated no significant changes in peripheral blood erythroid elements between flight and ground control rats. The nonadherent bone marrow on flight day 13 showed a lower number of recombinant rat interleukin-3 (rrIL-3)-responsive and rrIL-3 + rhEpo-responsive blast-forming unit erythroid (BFU-e) colonies in flight rats compared with ground control rats. On landing day, a slight increase in the number of rhEpo + rrIL-3-responsive BFU-e colonies of flight animals compared with ground control rats was evident. Nine days postflight, bone marrow from flight rats stimulated with rhEpo alone or with rhEpo + rrIL-3 showed an increase in the number of colony-forming unit erythroid colonies and a decrease in BFU-e colonies compared with ground control rats. This is the first time that animals were injected with rhEpo and subsequently blood and tissues were collected during the spaceflight to study the regulation of erythropoiesis in microgravity.

Quantitative and Qualitative Characteristics of Colony-Forming Unit-Erythroid Colonies in Myelodysplastic Syndrome Patients

The microcytofluorometrical method was applied to determine the relative hemoglobin (Hb) content in the bone marrow colony-forming unit-erythroid (CFU-E) colonies from 6 patients with myelodysplastic syndromes (MDS) and 10 healthy subjects. This method relies on a photochemical reaction, by which intracellular Hb is converted into fluorescent porphyrin using a 0.2 M mercaptoethylamine solution (an SH donor) and violet light (lambda = 405 nm). The relative Hb content was determined as a function of the intensity of emitted porphyrin fluorescence. The number of colonies identified by porphyrin fluorescence was smaller in MDS patients than in normal subjects. The relative Hb content was also lower in MDS patients than in normal subjects. In addition, the coefficient of variation of the relative Hb content in the CFU-E colonies was larger in MDS patients than in normal subjects. These findings suggest that colonies with low relative Hb content undergo impaired erythropoiesis and that the CFU-E colonies undergoing the impaired erythropoiesis are mixed with CFU-E colonies showing normal erythropoiesis in the bone marrow of MDS patients.

A microcytofluorometric method for quantitative and qualitative evaluation of CFU-E and BFU-E colonies

A new method has been developed for the precise identification of human bone marrow colony forming unit erythroid (CFU-E) and burst forming unit erythroid (BFU-E) colonies, and for determination of the hemoglobin contents using microcytofluorometry. The method relies on a photochemical reaction in which intracellular hemoglobin is converted into fluorescent porphyrin under violet light (lambda = 405 nm) in the presence of an SH-donor (mercaptoethylamine hydrochloride). The CFU-E and BFU-E colonies showed red fluorescence with two spectrum peaks at 600 and 650 nm when illuminated by violet light. These two peaks are consistent with those of porphyrin fluorescence. The porphyrin fluorescence was not inducible in colony forming unit granulocyte-macrophage (CFU-GM) colonies, while 20% of the CFU-GM colonies were false positive with respect to the conventional benzidine reaction. The photochemically inducible fluorescence began to appear in BFU-E colonies on the 4th day of culture, while the same colonies started to be positive for the benzidine reaction on the 9th day. Therefore, the photochemical reaction was more specific and sensitive than the benzidine reaction for the identification of CFU-E and BFU-E colonies. In addition, this method enabled us to measure the hemoglobin level in the cells forming the colonies because the intensity of the fluorescence was proportional to the amount of hemoglobin when the photochemical reaction was carried out for 50 min.(ABSTRACT TRUNCATED AT 250 WORDS)