Target cells for Friend virus-induced erythroid bursts in vitro

Erythropoietin (Epo) acts on mouse bone marrow cells in vitro in plasma clot or methyl cellulose culture systems to induce the formation of single erythroid colonies, or clusters of erythroid colonies termed bursts. Our laboratory has recently reported the observation that infection of mouse bone marrow cells in vitro with the polycythemia-inducing strain of Friend virus (FV) resulted in the formation of erythroid bursts after 5 days in plasma clot culture in the absence of added Epo. We have now used this system to characterize the target cells for this FV-induced erythroid transformation. The greatest number of FV bursts were observed when marrow cells were obtained from mice whose erythropoiesis had been stimulated by bleeding or phenylhydrazine treatment. Bleeding also resulted in an increase in the number of FV bursts following the infection of spleen cells in vitro. Hypertransfusion of mice, which results in decreased erythropoiesis, yielded a reduced number of FV bursts in vitro, as did prior treatment with actinomycin D. Cell separation studies using velocity sedimentation at unit gravity showed that the cells, which give rise to FV bursts, sedimented with a modal sedimentation velocity between 5.1–8.5 mm/hr. The Epo-dependent colony-forming unit erythroid (CFU-E), which gives rise to a single erythroid colony, also sediments with a modal velocity between 5.1–8.5 mm/hr, while the Epo-dependent day 8 burst-forming unit erythroid (day 8 BFU-E) sediments with a modal velocity between 3.0–6.0 mm/hr. A 20 min incubation of marrow cells with high specific activity ³H-thymidine, prior to virus infection, resulted in a 75–80% reduction in the number of FV bursts. Mixing cells from the upper portion of the gradient, which yielded no FV bursts, with cells from an area in which high numbers of FV bursts were observed did not result in the inhibition of burst formation. These experiments indicate that the primary target cells for FV bursts in vitro are most probably erythroid precursor cells that have matured beyond the day 8 BFU-E and are closely related to the CFU-E.     

Interleukine-17-induced inhibitory effect on late stage murine erythroid bone marrow progenitors

Recent studies have shown that the T cell-derived cytokine, interleukin-17 (IL-17), stimulates hematopoiesis, specifically granulopoiesis inducing expansion of committed and immature progenitors in bone marrow. Our previous results pointed to its role in erythropoiesis too, demonstrating significant stimulation of BFU-E and suppression of CFU-E growth in the bone marrow from normal mice. As different sensitivities of erythroid and myeloid progenitor cells to nitric oxide (NO) were found, we considered the possibility that the observed effects of IL-17 were mediated by NO. The effects of recombinant mouse IL-17, NO donor (sodium nitroprusside - SNP) and two NO synthases inhibitors (L-NAME and aminoguanidine) on erythroid progenitor cells growth, as well as the ability of IL-17 to induce nitric oxide production in murine bone marrow cells, were examined. In addition, we tested whether the inhibition of CFU-E colony formation by IL-17 could be corrected by erythropoietin (Epo), the principal regulator of erythropoiesis. We demonstrated that IL-17 can stimulate low level production of NO in murine bone marrow cells. Exogenously added NO inhibited CFU-E colony formation, whereas both L-NAME and aminoguanidine reversed the CFU-E suppression by IL-17 in a dose-dependent manner. The inhibition of CFU-E by IL-17 was also corrected by exposure to higher levels of Epo. The data obtained demonstrated that at least some of the IL-17 effects in bone marrow related to the inhibition of CFU-E, were mediated by NO generation. The fact that Epo also overcomes the inhibitory effect of IL-17 on CFU-E suggests the need for further research on their mutual relationship and co-signalling.     

Changes in hemopoietic and regulator levels in mice during fatal or nonfatal malarial infections. I. Erythropoietic populations

Erythroid precursors BFU-E and CFU-E and erythroblasts (ERB) were monitored in the marrow and spleen of mice during fatal or nonfatal malaria. Transient depletions of marrow CFU-E and ERB without modification of BFU-E or erythropoietin (Epo) levels were found as early events in fatal infections. Before anemia development, erythropoiesis was reduced in the bone marrow but increased in the spleen. During the anemic phase, for comparable levels of anemia, plasma Epo levels were elevated to a similar degree in fatal and nonfatal malaria. In the bone marrow, CFU-E increased twofold and BFU-E were usually reduced as expected in severe anemia. ERB populations increased but remained below or within normal values, suggesting an impairment of marrow erythropoiesis related to early events following infection. In contrast, in the spleen, ERB production was strongly simulated but amplification of ERB, CFU-E, and BFU-E populations was 2.5-fold lower in fatal than in nonfatal malaria. The results suggest that a defect in amplification of splenic erythropoiesis is a crucial determinant of the fatal outcome of malarial infection. This may have been mediated by a defective stem cell migration or multiplication. Some evidence obtained during recovery stages suggested that a factor(s) other than Epo may control splenic erythropoiesis during the anemia associated with malaria.     

Association between leukemic erythroid progenitors and bone marrow macrophages

Previous ultrastructural investigations have shown that the erythroblastic island is composed of erythroblasts at different stages of maturation which are intimately associated with a central macrophage. However, it is still unclear at which stage of erythroid differentiation this interaction occurs, mainly because of the lack of purified populations of normal erythroid progenitors [erythroid colony-forming units (CFU-E) and erythroid burst-forming units (BFU-E)] and early precursor cells (proerythroblasts) and because of our limited knowledge of their ultrastructural characteristics. In the present work we analyzed the ultrastructure of CFU-E enriched from normal human bone marrow by avidin-biotin immune rosetting and leukemic blasts of erythroid origin from two patients. Normal and leukemic CFU-Es were defined as glycophorin A (GPA)-negative blasts, devoid of rhopheocytosis, containing some ferritin molecules, either free in the cytoplasm or associated with theta-granules (theta-Gr) in the Golgi zone. Peroxidase activity was detected in the endoplasmic reticulum of these blasts. A preproerythroblast stage was identified, which corresponded to an intermediate phenotype with few GPA sites and rhopheocytosis. In contrast to hemoglobin synthesis, which was absolutely dependent on the presence of erythropoietin (Epo) during culture for 24 hours, ferritin molecules accumulated in the absence of Epo. Interestingly, leukemic CFU-E-like blasts were always in contact with bone marrow macrophages and adhesion between these cell types resisted mechanical dissociation. This result suggests that erythroid progenitors may be part of the erythroblastic island. The mechanisms involved in erythroblast-macrophage binding are still unknown, but the expression by macrophages and erythroid progenitors of receptors for fibronectin and thrombospondin (TSP), as well as their respective ligands in the case of macrophages, suggests that these molecules could be involved in the formation of the erythroblastic island.     

Myeloproliferative sarcoma virus: its effects on erythropoiesis in adult DBA/2J mice

Adult susceptible mice (DBA/2J) infected with MPSV (myeloproliferative sarcoma virus), a defective RNA tumour virus, develop splenomegaly and progressive disruption of the haematologic system culminating in death. The present study was specifically directed toward determining the effects of the virus on erythroid differentiation. Early and late precursor cells (erythroid burst-forming units; BFU-E and colony-forming units; CFU-E, respectively) were evaluated by the ability of bone marrow and spleen cells to form colonies of fully differentiated erythroid cells in vitro. MPSV caused substantial modification of both the BFU-E and CFU-E populations in the bone marrow and spleen of infected animals. Changes were detected in the CFU-E population preceding any significant increase in spleen weight. In the bone marrow, the proportion of CFU-E cells increased almost twofold by days 5-10 after virus infection but decreased by day 15. In the spleen, CFU-E frequency rose 40-fold by days 10-15 and then declined steadily prior to death. At the peak of CFU-E expansion, a small proportion of the population appeared to be erythropoietin (Ep) independent, although there was no evidence of a complete switch to Ep-independence which occurs in Friend virus-induced erythroleukemia. Dose-response curves showed that none of these data could be explained in terms of a changing responsiveness to Ep. However, evidence is presented that indicates that BFU-E from MPSV-infected animals lose or have a reduced requirement for burst-promoting activity (BPA) relative to normal cells although their progeny still need Ep for terminal erythroid differentiation.     

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.     

Developmental changes in plasma erythroid colony-stimulating activity in mice: cyclic erythropoiesis associated with rapid growth.

To establish a role of erythropoietin (Epo) in regulation of fetal and neonatal erythropoiesis, plasma erythroid colony-stimulating activity (ECSA) in developing mice was measured by an erythroid colony-forming assay using fetal mouse liver cells. The ECSA in fetal and neonatal plasmas showed dose-response curves parallel to standard Epo curve and additive effects with standard Epo on the colony formation. Most of the plasma ECSA was neutralized by an anti-Epo monoclonal antibody. These results suggest that the plasma ECSA detected by the present bioassay is predominantly due to Epo. On day 12-14 of gestation, the plasma ECSA levels were at the highest values; thereafter the levels oscillated up to the age of 4 weeks. The packed cell volume (PCV) also oscillated, but with the reverse phase. Oscillation in PCV was associated with the growth. There was an inverse relationship between plasma ECSA and PCV levels throughout the prenatal and early postnatal periods. The results indicate that erythropoiesis in fetal and neonatal mice is regulated mainly on the basis of PCV-ECSA feedback control mechanism.     

Survival of erythroid burst-forming units and erythroid colony-forming units in canine bone marrow cells exposed in vitro to 1 MeV neutron radiation or X rays

Conditioned media (CM) from allogeneic stimulated cultures of light density cells (less than 1.08 g/cm3) from the peripheral blood of normal dogs were used to stimulate the growth of erythroid burst-forming units (BFU-E) in bone marrow from normal dogs. Maximum numbers of BFU-E were obtained when 5% (vol/vol) 3 X CM and 2 U/ml erythropoietin were added to plasma clot cultures of bone marrow cells. In addition, the radiation sensitivity (D0 value) was determined for CFU-E and for BFU-E in bone marrow cells exposed in vitro to 1 MeV fission neutron radiation or 250 kVp X rays. BFU-E were more sensitive than CFU-E to the lethal effects of both types of radiation. For bone marrow cells exposed to 1 MeV neutron radiation, the D0 for CFU-E was 0.27 +/- 0.01 Gy, and the D0 for BFU-E was 0.16 +/- 0.03 Gy. D0 values for CFU-E and BFU-E were, respectively, 0.61 +/- 0.05 Gy and 0.26 +/- 0.09 Gy for cells exposed to X rays. The neutron RBE values for the culture conditions described were 2.3 +/- 0.01 for CFU-E and 1.6 +/- 0.40 for BFU-E.     

Zidovudine (AZT) treatment suppresses granulocyte-monocyte colony stimulating factor receptor type alpha (GM-CSFR alpha) gene expression in murine bone marrow cells

In vitro exposure of murine bone marrow cells to increasing concentrations of zidovudine (AZT, 0.1-50 microM) had a concentration dependent suppressive effect on the growth of granulocyte-monocyte colony forming unit (CFU-GM) derived colonies. In our previous published study, the mechanism of AZT-induced suppression of erythroid colony forming unit (CFU-E) derived colonies was linked to a decrease in erythropoitin receptor (Epo-R) gene expression. In this study, we have observed that AZT exposure also induced a concentration dependent suppressive effect (35-90%) on GM-CSF receptor type alpha (GM-CSFR alpha) gene expression. The suppression of GM-CSFR alpha mRNA expression was specific, since AZT caused a much lower decrease (15-22%) on the IL-3 receptor type alpha (IL-3R alpha) message level, and had an insignificant effect on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and c-myc message levels. Erythropoietin (Epo) therapy has been used for reversal of AZT induced erythroid toxicity. Exposure to increasing concentrations (10-500 U/ml) of GM-CSF was unable to override the suppressive effect of AZT on CFU-GM derived colonies, however, treatment in combination with IL-3 (10-250 U/ml) ameliorated the suppressive effects of AZT on CFU-GM and on GM-CSFR alpha and IL-3R alpha gene expression. These findings suggest a mechanism via which AZT may suppress granulocyte-monocyte specific differentiation in murine bone marrow cells. These data also suggest that a combination of GM-CSF and IL-3 may be a superior therapeutic intervention for AZT-induced neutropenia.     

Hemoglobin switching in sheep and goats. VI. Commitment of erythroid colony-forming cells to the synthesis of betaC globin

Bone marrow from mature goats and sheep was cultured in plasma clots, and three erythropoietin (ESF)-dependent responses-growth (colony formation), differentiation (globin production), and initiation of hemoglobin C (alpha2beta2C) synthesis--were quantitated. ESF concentrations below 0.01 U/ml supported colony growth and adult hemoglobin production in cultures of goat marrow, while maximal hemoglobin C synthesis (70%), as measured between 72 and 96 h in culture, required a 100-fold higher ESF concentration. Sheep marrow was cultured in a medium enriched to enhance growth and to permit complete maturation of colonies. These colonies active in hemoglobin synthesis between 24 and 96 h produced mainly adult hemoglobin, and only between 96 and 120 h did sheep colonies develop which produced mainly hemoglobin C (up to 70%). A similar heterogeneity may exist among goat colonies. Thus, when goat bone marrow was fractionated by unit gravity sedimentation, more hemoglobin C synthesis was observed in colonies derived from cells of intermediate sedimentation velocity than in colonies derived from the most rapidly sedimenting cells. Brief exposure of sheep (in vivo) and goat (in vitro) bone marrow to a high ESF concentration committed precursor cells to the generation of colonies which, even at low ESF concentration, produced hemoglobin C. Committment to hemoglobin phenotype appears to be an early and probably irreversible event in the development of an erythroid cell.     

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

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

A novel way to induce erythroid progenitor self renewal: cooperation of c-Kit with the erythropoietin receptor

Red blood cells are of vital importance for oxygen transport in vertebrates. Thus, their formation during development and homeostasis requires tight control of both progenitor proliferation and terminal red cell differentiation. Self renewal (i.e. long-term proliferation without differentiation) of committed erythroid progenitors has recently been shown to contribute to this regulation. Avian erythroid progenitors expressing the EGF receptor/c-ErbB (SCF/TGFalpha progenitors) can be induced to long-term proliferation by the c-ErbB ligand transforming growth factor alpha and the steroids estradiol and dexamethasone. These progenitors have not yet been described in mammals and their factor requirements are untypical for adult erythroid progenitors. Here we describe a second, distinct type of erythroid progenitor (EpoR progenitors) which can be established from freshly isolated bone marrow and is induced to self renew by ligands relevant for erythropoiesis, i.e. erythropoietin, stem cell factor, the ligand for c-Kit and the glucocorticoid receptor ligand dexamethasone. Limiting dilution cloning indicates that these EpoR progenitors are derived from normal BFU-E/CFU-E. For a detailed study, mEpoR progenitors were generated by retroviral expression of the murine Epo receptor in bone marrow erythroblasts. These progenitors carry out the normal erythroid differentiation program in recombinant differentiation factors only. We show that mEpoR progenitors are more mature than SCF/TGFalpha progenitors and also do no longer respond to transforming growth factor alpha and estradiol. In contrast they are now highly sensitive to low levels of thyroid hormone, facilitating their terminal maturation into erythrocytes.     

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.     

An erythropoietic stimulating factor similar to erythropoietin released by macrophages after treatment with silica

An erythropoietic stimulating factor (ESF) can be detected in the supernatant from fetal liver and adult bone marrow and spleen cells when preincubated with the macrophage-specific cytotoxic agent, silica. Stimulation is observed in 12-day fetal liver CFU-E cultures in the absence of added erythropoietin (Ep). The concentration of ESF in the supernatant added to CFU-E cultures is dependent on the preincubated cell dose and the volume added. The stimulating activity is abolished when mice are hypertransfused and increased above normal values when mice are bled. A concentrated silica-treated spleen supernatant was able to stimulate erythropoiesis in the polycythemic mouse bioassay. It is concluded that the ESF is similar, if not identical, to Ep.     

Segregation of mouse hemopoietic progenitor cells using the monoclonal antibody,YBM/42

A rat monoclonal antibody, YBM/42, directed against mouse leukocyte common antigen, was used for the analysis and separation of hemopoietic progenitor cells from mouse bone marrow and fetal liver. Cells were fractionated on a FACS-II cell sorter and the resulting subpopulations examined for their morphology and ability to form colonies in agar (for day 7 colonies) and methylcellulose (for day 2 erythroid clones). The antibody bound to all leukocytes, including blast cells and day 7 hemopoietic progenitor cells (day 7 colony forming cells, CFC), but not to erythrocytes or nucleated erythroid cells. This antibody can be used to advantage to enrich for early progenitor cells from mouse fetal liver, in which the majority of cells (70%) are nucleated erythroid cells. In day 12 fetal liver, approximately 10% of all cells bind this antibody strongly and, of these approximately 70% are blast cells. Contained within this positive population are 95% of all day 7 CFC. In the most enriched fraction about 20% of the cells formed day 7 colonies. This represents a 25-fold enrichment over unsorted fetal liver. The negative fractions contain 94% of all cells forming erythroid clones (≥8 cells) on day 2 of culture (day 2 CFU-E). In the most enriched fraction, 20% of the cells are day 2 CFU-E. Day 7 CFC can therefore be well separated from day 2 CFU-E, with good recovery of both cell types, by use of a single label. Day 7 colony forming cells were classified as granulocyte (G-CFC), macrophage (M-CFC), mixed granulocyte/macrophage (GM-CFC), pure erythroid (E), or mixed erythroid (E_mix). A high enrichment for multipotential cells is achieved and constitues 3–5% of cells in the most enriched fraction. Most types of day 7 CFC could not be separated with YMB/42, but GM-CFC and M-CFC exhibit a broader distribution than the other CFC with regard to fluorescence intensity. This implicit heterogeneity in GM-CFC and M-CFC is further substantiated by the finding that myeloid progenitors in the different FACS fractions also share a differential reactivity to different sources of growth factors.     

Effects of beta adrenergic agents and prostaglandin E1 on erythroid colony (CFU-E) growth and cyclic AMP formation in Friend erythroleukemic cells

The formation of erythroid colonies from bone marrow and spleen cells infected with the polycythemic strain of the Friend virus (FV-P) was characterized in an in vitro methyl cellulose colony-forming system in response to prostaglandin E1 and the beta-2 adrenergic agonist, albuterol. Both drugs markedly inhibited the formation of CFU-E colonies of FV-P-infected bone marrow and spleen in the absence or presence of erythropoietin. The albuterol-mediated inhibition of CFU-E colonies (FV-P-infected) was selectively blocked by butoxamine, a beta-2 antagonist. Adenylate cyclase (AC) activity was also determined in FV-P spleen membrane preparations in response to albuterol and PGE1. Both agents stimulated enzyme activity, and butoxamine blocked the stimulation seen with albuterol. The ability of albuterol and PGE1 to stimulate AC activity in the FV-P-infected cells suggests that the effects of these agents on CFU-E formation may be mediated by specific beta-2 adrenergic and PG receptors through the adenylate cyclase-cyclic AMP system.     

Erythroid expansion and survival in response to acute anemia stress: the role of EPO receptor, GATA-1, Bcl-xL and caspase-3

Erythropoietic stress occurs under conditions of tissular hypoxia, such as anemia. Functional relationships between erythroid bone marrow (BM) proliferation, differentiation, the expression of survival and apoptotic related proteins, as well as the features of the BM microenvironment upon acute anemic stress, are not fully elucidated. To achieve this aim, CF-1 Swiss mice were injected with a single dose of 5-fluorouracil (5-FU, 150 mg/kg ip) and a multiparametric analysis was conducted for 20 days. Apoptosis (TUNEL assay), BM architecture organization (scanning electronic microscopy), proliferation (DNA assay), differentiation (clonogenic cultures), expression of survival erythroid related proteins (EPO-R, GATA-1, Bcl-xL) as well as the expression of apoptotic- related proteins (Bax, activated Caspase-3) by Western blotting, were evaluated. Experimental data showed that apoptosis, arrest of cell proliferation and disruptions of BM architecture were maximal within the first period of acute stress (1-3 days). Bax and caspase-3 overexpressions were also coincident during this acute period. Moreover, from day 5 upon drug challenge BM responds to acute stress through the EPO-EPO-R system, prompting expressions of GATA-1 and Bcl-xL. Erythroid proliferation rates and red-cell-committed progenitors enhanced in a coordinated way to restore the size and function of the red cell compartment. A second overexpression wave of active caspase-3 was noticed during stress recovery. Together, these results indicate that in response to acute stress a dramatic increase in CFU-E (erythroid colony forming units) population is concomitant with upregulation of EPO-R, GATA-1 and Bcl-xL in the BM erythroid compartment, and that these concurrent processes are crucial for acquiring proper erythroid cell functionality without delayed response to tissular hypoxia.     

A model of hemopoietic stress in a lactate dehydrogenase mouse mutant with hemolytic anemia

A codominantly inherited mutation of the lactate dehydrogenase (LDH) in the C3H mouse causes a severe hemolytic anemia in homozygous mutants, whereas viability and fertility are close to normal. Investigation of multipotent hemopoietic stem cells (CFU-S), myeloid (GM-CFC) and erythroid progenitors (BFU-E, CFU-E) in femur and spleen indicates a general shift from bone marrow to splenic hemopoiesis. In terms of total body hemopoiesis, however, the BFU-E pool is 1.4- and the CFU-E pool 19-fold enlarged, whereas CFU-S and GM-CFC show little or no deviation from normal. It is concluded that this mouse mutant is an appropriate model of long-term hemopoietic stress showing that compensation in this severe hemolytic anemia is achieved primarily by an increase of the number of the most mature erythroid progenitors.     

Usefulness of the assessment of erythroid progenitors growth for differential diagnosis of primary and secondary polycythemias

Erythropoietic progenitors from bone marrow of patients with polycythemia vera (PV), secondary polycythemia (SP) and healthy subjects (HS) were cultured in plasma clot diffusion chambers in vivo. The chambers were inserted into the peritoneal cavities of rats, which 24 and 2 h before implantation received an injection of phenylohydrazine. Control experiments were done without erythropoietin (Epo) stimulation. Colonies after 2 and 7 days of culture were considered to be formed by mature erythropoietic progenitors (CFU-D-E) and burst forming cells (BFU-D-E), respectively. PV-erythroid progenitors, both BFU-D-E and CFU-D-E produced markedly more colonies than those from SP and HS, especially in experiments without Epo stimulation (p less than 0.01). The plating efficiency in SP was comparable to that noted in HS (p greater than 0.05). These results have led us to postulate that the study of erythroid progenitor clonal proliferation in plasma clot diffusion chamber can be helpful in the differential diagnosis of PV and SP, when other clinical and laboratory findings are not sufficiently convincing.