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Summary. Erythroid colonies derived from the circulating early erythroid precursor (BFU-E) of a patient with congenital dyserythropoietic anaemia type I (CDA I) have been grown in plasma clot and studied by electron microscopy. The number of circulating BFU-E was in the normal range with a roughly normal appearance at the light microscopic level. However, investigation of individual colonies by electron microscopy has always shown a mixture of normal and abnormal erythroblasts exhibiting the typical nuclear aberrations found in vivo. The proportion of normal erythroblasts varied from one colony to another. After the release of the cells from the clot in order to permit new cellular interactions, macrophages were observed to phagocytose abnormal erythroblasts but also a few erythroblasts with normal nuclei. These findings demonstrate that CDA I is a disorder which results from a defective erythroid stem cell but that the progeny of each BFU-E may vary considerably in the extent to which they express the morphological defects. Based on studies of cultures of BFU-E, similar conclusions were previously made for CDA 11.

We studied monoaminergic mechanisms of regulation of the pool of erythropoietic precursors in active and passive mice under conditions of conflict situation and during paradoxical sleep deprivation. It was found that in animals with different behavior subjected to experimental neuroses the regulatory effects of CNS monoamines on proliferation and differentiation are mediated via adrenergic and erythropoietin-sensitive structures on erythroid stem cells. The revealed intergroup differences in precursor proliferation and differentiation rates (and erythrokaryocyte content in the bone marrow) are related to peculiarities in the activity of monoaminergic system and their interaction with peripheral structures on erythropoietic precursors.

The flow cytometer-based micronucleus assay was used to study the effects on chromosomes in erythroid cells of CBA/Ca mice after extended exposure to 50 Hz magnetic field (MF), 14 μT, peak-to-peak (p-p). The study included two different experiments: (a) mice exposed in utero during 18 days of their prenatal stage, and (b) adult mice exposed for 18 days. In experiment (a) 35 days after exposure was terminated, peripheral blood was drawn from the mice exposed in utero to determine whether the exposure had a genotoxic effect on the pluripotent erythroid stem cells. About 200000 polychromatic erythrocytes (PCE) and 200000 normochromatic erythrocytes (NCE) were analysed from each of 20 exposed mice. The EMF exposure did not significantly change the frequency of micronucleated PCE or NCE in comparison with 20 sham-irradiated mice. There was no difference in the proportion of PCE between exposed and unexposed animals. Similarly, in experiment (b) no differences were seen between EMF exposed and unexposed adult mice when samples of peripheral blood were taken at the end of exposure and analyzed for micronuclei in PCE and NCE. The proportion of PCE was the same in both groups. The results indicate that exposure to EMF does not induce direct or indirect effects on chromosomes in erythroid cells expressed as increased levels of micronucleated erythrocytes of mice. No indications of delayed genetic effects were found. Bioelectromagnetics 22:351–357, 2001. © 2001 Wiley-Liss, Inc.

A survey of glycophorin A (gpa) in vivo somatic cell mutation in a population of 394 healthy people from 8 to 77 years of age (mean age +/- SD 41 +/- 15 years) revealed a subset of 37 individuals with stably elevated allele-loss and/or allele-loss with duplication variant erythrocyte frequencies (Vf) exceeding 30 x 10(-6). These 37 individuals with gpa outlier Vf are significantly older (P0.001) than the remainder of the larger study population from which they were drawn reflecting a highly significant increase in the prevalence of these individuals in the population beyond age 40 years. A study of hpt mutant frequencies (Mf) in the peripheral blood T-lymphocytes of 27 of these individuals, together with 15 matched control individuals with unremarkable gpa Vf, was undertaken to determine if these subjects also displayed elevated mutation frequencies at this independent locus indicative of globally elevated somatic mutation. The hprt Mf in these 27 subjects (geometric mean 11.5 x 10(-6)(dispersion interval 5.8 x 10(-6) to 22.8 x 10(-6)) was not significantly different from that observed in the 15 controls (geometric mean 12.1 x 10(-6)(dispersion interval 5.7 x 10(-6) to 25.5 x 10(-6)). These Mf are higher than typically reported values reflecting the older age distribution of these individuals (arithmetic mean age +/- SD 53 +/- 12 and 50 +/- 16 years for the subjects and controls, respectively). Taken together, these data suggest that several genetic mechanisms may be responsible for producing the gpa outlier Vf observed in these subjects. The observation that hprt Mf were not increased indicates that the majority did not arise by a genome-wide increased rate of somatic mutation detectable at both loci. The fixation and subsequent expansion of 'jackpot' mutations at the gpa locus occurring early in embryonic/fetal development also does not appear to be a predominant mechanism. Some cases may result from a stable over-representation of gpa variant cells, perhaps associated with a marked age-dependent decrease in the number of contributing erythroid stem cells in the bone marrow. The subset that displays elevated allele-loss with duplication Vf involving both gpa alleles may represent individuals with increased rates of somatic recombination. Elevations arising by this mechanism are not detected in the hprt assay, but could be confirmed using a autosomal locus in vivo somatic cell mutation endpoint such as the hla-a assay. Of primary biological significance, these results demonstrate that genetics/stochastic processes leading to the loss of heterozygosity of somatic cells occur ubiquitously in humans and in some individuals this level of somatic mosaicism can approach a frequency of 10(-3) at the gpa locus in erythroid lineage cells.

The glycophorin A (GPA) mutation assay was used to examine the risk of in vivo somatic mutation in infants following neonatal administration of vitamin K. The assay assesses damage to erythroid stem cells by measuring the frequency of NO and NN variant red cells of MN blood group heterozygotes using FACS analysis. Blood samples were obtained from 178 infants aged between 10 and 183 days. Twenty-six children were excluded from study having received a blood transfusion. Sixty-four of the remaining 152 infants were of the MN phenotype, samples from whom were analysed using the assay system, providing the first data of NO and NN variant frequencies (vfs) in children aged less than 1 year. Twenty of these 64 infants received vitamin K orally (group A), 17 intramuscularity (group B) and 25 intravenously (group C). Results were compared with those from a reference population of children aged 1–15 years. There were no significant differences in NO, NN and total vf between any of groups A, B and C. For all groups both NO and total vf were significantly lower than those for the control population. This result is of some interest and clearly warrants further investigation. NN and total vfs were greater than the 95th percentile for the pooled data from groups A, B and C in three instances, one in each group. It was thus not possible to demonstrate an association between the route of vitamin K administration and an increase in mutation at the GPA locus.

Erythroid stem cell proliferation is regulated by lymphokines and erythropoietin. The helper subset of T lymphocytes is known to produce the erythroid growth factor IL-3 or burst-promoting activity (BPA), while the suppressor subset seems to inhibit the erythroid growth. Leukocyte-conditioned media derived from white cells of nonanemic elderly were reported to provide defective support to the erythropoiesis. In two groups of elderly, nonanemic and anemic, we studied the ability of T lymphocytes to stimulate the BFU-E growth and the in vitro effect of cimetidine, as a drug that inhibits the suppressor T lymphocytes. Culture data were then compared with the peripheral blood lymphocyte picture. The study shows that defective mononuclear cell support to the BFU-E growth, namely due to reduced absolute number of the T4 subset of T lymphocytes, can be observed in both anemic and nonanemic elderly. It is suggested that isolated defective BPA production is not always sufficient to induce anemia. In most cases, anemia of unexplained origin in senescence would be due to the concomitance of both BFU-E impairment and defective BPA production. The simultaneous evaluation of BFU-E growth, lymphokine production, and the T-lymphocyte blood picture offers the best way to investigate the erythropoiesis of the elderly.

Techniques are described for the culture of multipotential, primitive, and mature mammalian erythroid colony-forming stem cells obtained from bone marrow, spleen, blood, or fetal tissues in semisolid cultures under defined, serum-deprived growth conditions. Inasmuch as colony formation is critically dependent on the presence of tissue-specific growth factors, the addition of purified recombinant interleukin-3 and erythropoietin affords the opportunity to examine the specific growth requirements of undifferentiated hematopoietic cells in a critical and quantitative manner through terminal differentiation for extended periods of time.

A 93-year-old woman presented with profound anemia (hematocrit 23% [0.23]); there was clumping of her red cells in test tubes and on peripheral blood smears. There was also a marked decrease in erythroid precursors in the bone marrow and reticulocytopenia in the peripheral blood. An IgM kappa monoclonal gammopathy was found in low concentration (approximately 1%) in her serum, and the cold agglutinins had a titer of 2560. However, the cold agglutinin titer of the supernatant after cryoglobulin precipitation was 40. Redissolving the cryoglobulin in the supernatant resulted in a cold agglutinin titer of 1280. Moreover, the addition of the patient's whole serum inhibited erythroid colony formation in culture. The inhibition was removed by cryoprecipitation of the cryoglobulin. The patient was given steroid therapy, to which she responded with reticulocytosis and an elevation of hematocrit. By 3 months, the cold agglutinin titer had fallen to 10. She remained well 4 years later. Whereas reports of cryoprecipitable cold agglutinins are rare, this case is unique because there have been no previous reports that these cold active proteins also have erythroid stem cell-suppressant properties.

Female C57BL/6X DBA/2 hybrid mice were exposed to two concentrations of styrene in inhalation chambers for 6 h/day, 5 days per week. The concentrations were 220 ppm for a period of up to 8 weeks and 440 ppm for 2 days, followed by 330 ppm for up to 8 weeks due to early deaths after 440 ppm. Parallel groups were also treated with 5% ethanol in the drinking water during the days of styrene exposure. In the peripheral blood only a lymphocytopenia was found. The numbers of pluripotent haemopoietic stem cells, CFU-S, were unaffected, as were granuloid committed stem cells, CFU-C. Early and late erythroid stem cell numbers, BFU-E and CFU-E, showed high variability. At 220 ppm about half of the animals had CFU-E numbers below 80% of controls, at 440/330 ppm about two-thirds of the animals had BFU-E and CFU-E numbers below this level. An additional effect of ethanol was not found.

The numerical and morphological findings of erythroid burst colonies from the peripheral blood and bone marrow of two patients with congenital dyserythropoietic anemia type II (CDA-II) are described. In both patients there was an increase of medullary and peripheral BFU-E that was explained by a compensating mechanism against the destruction of erythrocytes. In most of the colonies normal and abnormal erythroblasts co-existed. The ultrastructural analysis of erythroblasts showed, as in vivo, bi- and multinuclearity, autophagic vacuoles, and aberrant membranes that sometimes gave rise to the double-membrane appearance. These abnormalities were found in both patients simultaneously in the blood and bone marrow. These findings point to defective erythroid stem cells. The clinical expression of the disease may depend partly on the ratio of normal to abnormal erythroid colonies.

Survivors of childhood acute lymphoblastic leukaemia (ALL) have a higher than expected risk of developing secondary acute myeloid leukaemia (AML). The glycophorin A (GPA) mutation assay measures the frequency of variant NO and NN erythrocytes in MN heterozygotes. A raised variant frequency (Vf) has been shown in patients treated with chemotherapy known to be at risk of secondary leukaemia. ALL patients were investigated for increased Vf using the GPA assay. Vf's at diagnosis were not significantly different from controls (NO Vf P = 0.193 ; NN Vf P = 0.790). During treatment Vf's increased significantly (NO Vf P = 0.001 ; NN Vf P = 0.001). NO Vf returned to control values (P = 0.169) within 5 years from diagnosis but NN Vf remained significantly raised (P = 0.014). Three study patients developed secondary AML. At diagnosis of AML all three had significantly increased Vf. The first had a significantly raised Vf at routine follow-up 19 years following diagnosis of ALL then developed AML 3.5 years later. The second had a significantly raised NN Vf at diagnosis of ALL indicating possibly prior exposure to a mutagen or defective DNA repair involving erythroid stem cells. We conclude that a raised Vf detected by the GPA assay can act as a marker for the development of secondary induced leukaemia and can be used to screen individuals at a known high risk of this complication.

Introduction Effective therapies for Sickle Cell Anaemia (Hb SS) reduce blood concentration of HbS by transplanting erythroid stem cells or altering globin gene expression with hydroxyurea. A relatively stable reduction in HbS concentration intermediate between these approaches (10–45% HbS) can also be achieved by regular automated red cell exchange transfusion (RBCex), but concerns about cost-effectiveness, alloimmunisation, iron load, pathogen exposure and vascular trauma have limited the use of this treatment modality. Methodology A retrospective study of sixteen fully counselled HbSS individuals with serious complications undergoing regular RBCex with partially phenotype-selected, leucodepleted red cells for a median of 36 (range 11–65) months, for whom accurate pre- and post- RBCex admission data was available. The number of episodes of sickle-related hospitalisation and total inpatient days sustained per year by the group before enrolment in RBCex was compared to the number on RBCex. Excluded from analysis were patients having single RBCex for surgery, pregnancy, or non-recurrent acute complications, having manual exchange transfusions or unable to comply with regular RBCex. Four patients from other units were excluded because complete data on previous admissions was lacking. Target post-procedure haemoglobin was 10–11gm/dL (PCV = 0.31–33) and HbS% Results Between September 1995 and July 2003, 547 RBCex procedures were performed on 56 patients (age: range 16–52 yrs, median 31.5 yrs, mean 29.7 years). Forty patients were excluded from analysis for reasons above. Analysed patients underwent a mean of 7.71 RBCex procedures per year (range 5.12–9.89, median 7.49). A mean of 7.8 (range 4.25–10, median 8) donor red cell units were transfused per procedure. RBCex was performed at a mean of 6.97-week intervals (range 5.26–10.16, median 6.91). Eleven patients (62.5%) required ‘in-out’ femoral vein Vascath insertion. Apheresis nurses placed all catheters in a day-care setting. In the five years before entering the program the patients experienced a mean of 2.03 inpatient episodes per year (range 0.4–5.2, median 1.6): on RBCex this fell to 1.02 per year [range 0–5.3, median 0.33 (p = 0.004)]. Inpatient days fell from a mean of 16.56 per year (range 2–40.6, median 11) to 8.27 [range 0–56.33, median 1.33 (p=0.007)] saving 208.87 in-patient bed days per year. The number of donor red cell units patients received increased from a mean of 15.15 per year (range 0.8–41.1, median 14.1) to 57.9 units per year [range 24–89.7, median 58.0 (p=0.004)]. No patient received any form of iron chelation. No increase in serum ferritin (p=0.796), or loss of vascular access sites, was identified. Two patients acquired new red cell alloantibodies (1-anti Kpa + anti-E; 1- non specific) during RBCex. Three already had alloantibodies (1 anti-e; 1 anti-Kpa + anti-C; 1 non-specific), but did not acquire any more while on RBCex. The presence of antibodies did not preclude continuing RBCex. No transfusion-related infections occurred. One patient died from unrelated causes at home. One novel phenomenon was identified: while on RBCex, ‘rebound’ crises tend to occur when HbS crosses the 45% threshold, emphasising the need for consistency using this therapy. Conclusion We contend that regular RBCex is effective therapy for carefully (self)-selected patients who severely symptomatic. It should be considered in those unresponsive to, or unwilling to take, hydroxyurea. Multidisciplinary support by a skilled team is essential.

Cartilage-hair hypoplasia (CHH) is an autosomal recessive metaphyseal chondrodysplasia which results in disproportionate short stature (mean adult height of 120-130 cm), hypoplastic hair and impaired cellular immunity. The defect has been assigned to chromosome 9p but the causative gene remains uncharacterized. We have previously shown that defective erythropoiesis is an integral part of CHH. The patients often present with anemia and macrocytosis. Irrespective of the degree of anemia, bone marrow cultures show impaired growth of the erythroid stem cells. Since growth hormone may regulate both body growth and erythropoiesis we have studied hematological characteristics and their correlations with growth and related parameters in 13 patients with CHH.

Thorotrast, a colloidal suspension of the long-lived radionuclide, thorium-232, was widely used as a radiographic contrast medium for several decades. Due to the poor excretion of the sol, however, Thorotrast would deposit in the liver, bone marrow and other tissue, and patients would receive alpha-particle irradiation for life. To gauge the cumulative genetic damage to hematopoietic stem cells due to chronic exposure to alpha particles, we conducted a multi-end-point evaluation in a 72-year-old man who had been administered a 32-ml bolus of Thorotrast during cerebral angiography performed over 40 years ago in 1950. Peripheral T lymphocytes were cultured to quantify the frequencies and cellular distributions of asymmetrical and symmetrical types of chromosome aberrations in first-division metaphases and micronuclei in cytokinesis-arrested interphase II cells. Aberrations were scored using classical chromosome group analysis methods and chromosome painting techniques. Assays of glycophorin-A (GPA) mutations in red blood cells were also performed to obtain a relative measurement of damage sustained by the erythroid stem cell population. Results revealed that approximately 30% of the lymphocytes in this patient contained one or more chromosome aberrations, the majority of which were of the "stable" type. About one-third of the lymphocytes with chromosome damage carried multiple aberrations, suggesting that significant numbers of stem cells survive exposures to alpha-particle radiation that induce complex genomic alterations. Increased frequencies of GPA mutations were observed, demonstrating that genomic damage is also induced in erythroid progenitors. The numbers of micronuclei in lymphocytes were only moderately increased compared to expected values for persons of comparable age, and thus this end point was not useful for quantifying exposure level. Despite the relatively severe burden of somatic cell damage induced by 40 years of internal alpha-particle irradiation, the patient remains surprisingly free of any serious illness.

The reactor accident at Chernobyl in 1986 necessitated a massive environmental cleanup that involved over 600,000 workers from all 15 Republics of the former Soviet Union. To determine whether the whole-body radiation received by workers in the course of these decontamination activities resulted in a detectable biological response, over 1,500 blood samples were obtained from cleanup workers sent from two Baltic countries, Estonia and Latvia. Here we report the results of studies of biodosimetry using the glycophorin A (GPA) locus in vivo somatic cell mutation assay applied to 734 blood samples from these workers, to 51 control samples from unexposed Baltic populations and to 94 samples from historical U.S. controls. The data reveal inconsistent evidence that the protracted radiation exposures received by these workers resulted in a significant dose-associated increase in GPA locus mutations compared with the controls. Taken together, these data suggest that the average radiation exposure to these workers does not greatly exceed 10 cGy, the minimum levels at which radiation effects might be detectable by the assay. Although the protracted nature of the exposure may have reduced the efficiency of induction of GPA locus mutations, it is likely that the estimated physical doses for these cleanup worker populations (median reported dose 9.5 cGy) were too low to result in radiation damage to erythroid stem cells that can be detected reliably by this method.

Lymphocyte development and ontogenetic changes in erythroid cells have been studied in chick-chick yolk-sac-embryo chimeras differing at the B locus antigens. Erythroid cells derived from the yolk sac or from the intraembryonic mesenchyme were demonstrated by indirect immunofluorescence in the peripheral blood of these allogenic chimeras. At 7 days of incubation, yolk-sac-derived red cells represent a majority in the peripheral blood. From 9 days of incubation onwards, embryo-derived erythrocytes appear in increasing proportions, making up approximately 90% of the peripheral blood cells at 17–18 days of development. After hatching, no yolk-sac-derived erythrocytes are found in the peripheral blood. Such a change from the yolk-sac-derived cells into embryo-derived cells was not observed in the lymphocytes, as analyzed using specific anti-B and anti-la antisera for detection of thymus and bursa cells, respectively. Ia-like antigens were detected on bursa cells using a triple layer immunofluorescence system. These results obtained from the allogeneic chimeras indicate that the early chicken yolk sac produces only transiently erythroid stem cells, while intraembryonic stem cells are involved in the production of definitive erythrocytes as well as of lymphocytes, both of T and B cells.

The erythropoietic stem cell compartment was studied in Friend-virus (polycythemic strain, FV-P) infected DBA/2 and NMRI mice with the CFUE and BFUE technique. Early after infection there was a depression in CFUE number in bone marrow and spleen, followed by an increase of the CFUE concentration, earlier and more pronounced in the spleen than in the marrow. Three days after FV-P infection an erythropoietin (Ep) independent CFUE population started to grow and replaced the normal Ep-dependent population within 8 to 12 days. The shift to Ep independency was not gradual. CFUE colonies of FV-P infected bone marrow cells were two to three times larger than control colonies after three days in vitro incubation. BFUE colonies increased in number during the first days of infection, but were totally lost after more than ten days. After velocity sedimentation of bone marrow cells of FV-P infected animals, however, the BFUE containing fractions showed normal BFUE colony growth and normal Ep sensitivity. In unfractionated bone marrow cell cultures BFUE colony growth could be observed later than ten days post infection when the cultures were refed with medium. It was therefore concluded that the loss of BFUE colony growth after FV-P infection was an in vitro artefact due to inadequate culture conditions.

Hemoglobin ontogenic changes have been studied in chimeras composed of a quail embryo grafted on a chick yolk sac. From the blood of such chimeras, quail erythrocytes are separated from chick by means of a specific immune hemolytic technique, thus yielding two populations of red cells, one deriving from yolk sac stem cells and the other from intraembryonic stem cells. Both populations comprise in succession primitive and definitive erythrocytes. However, quail primitive erythrocytes are produced in very small or often undetectable quantities, whereas quail definitive erythrocyte inflow increases steadily; on the other hand, chick cells make up the bulk of the primitive series and taper off in the definitive series. Previous work by our group has shown that intraembryonic stem cells travel to the yolk sac during the second phase of erythropoiesis. The results mean that the sequence of erythrocytic series is determined by a time program rather than by the origin of stem cells or site of erythropoiesis. All areas of the blastoderm may give off erythroid stem cells with similar potentialities, but most yolk sac stem cells engage in erythropoiesis at the early phase, while most intraembryonic stem cells do so later.

The concept that has emerged from our experiments and those of others is that erythroid stem cells are committed to undergo a program of erythroid differentiation with respect to the ultimate hemoglobin phenotype of their progeny erythrocytes. A clear distinction can be drawn between the switch from Hb A (or Hb F) to Hb C in sheep and the switch from Hb F to adult hemoglobin in humans. The former appears to be regulated in a relatively late erythroid stem cell with characteristics of CFU-E. In contrast, the CFU-E found in adult sheep bone marrow from animals that lack the beta C gene appear to be preprogrammed to produce only adult hemoglobin. Fetal stem cells may be induced to synthesize Hb C within a time frame that is similar to that seen in cultures of adult bone marrow. Thus, a common mechanism modulating the potential for expression of this gene and commitment of erythroid stem cells with respect to Hb C production in progeny erythroblasts seems quite likely. Again fetal CFU-E and BFU-E in animals lacking the beta C gene appear to be, for the most part, committed toward producing erythroblasts making Hb F. Further analysis will be required to determine at exactly which stage of stem cell differentiation this programming occurs and also the factors that are important in modulating the potential for fetal and adult hemoglobin synthesis.

Heterologous antisera to mouse brain tissue have activity against mouse pluripotent hemopoietic stem cells (CFU-S), but not against granuloid/macrophage committed precursor cells (CFU-C). In these studies we show that anti-mouse brain serum raised in a rabbit does not possess specific activity in vitro against late committed erythroid stem cells (CFU-E).

Summary. A concise review of recent advances in studies of mechanisms controlling the rate of erythropoiesis, including some original results, is presented. The first and second sections are focused on erythropoietin (Ep) and the erythroid stem cell respectively. In the third one, clinical conditions caused by a defect in the regulation of erythropoiesis are discussed.

The expression of fetal hemoglobin, Hb F, in the adult cells is cellularly restricted both in vivo and in culture. Because, in cultures of erythroid progenitors, subclones that express or fail to express Hb F are derived from the same erythroid stem cell, a mechanism must exist whereby Hb F expression segregates in the progeny of erythroid progenitors during their differentiation. We present mathematical analyses of experimental data which suggest that expression of Hb F in human adult erythroid cells occurs on a stochastic basis. We quantified Hb F expression among the subclones of erythroid bursts (clones) in vitro by labeling subclones with fluorescent anti-Hb F antibodies. The observed data were compared with predictions from a stochastic model with the assumption that the expression of Hb F in a subclone occurs with a probability P equal to the frequency of Hb F-expressing subclones in an experiment. There was good fit between the observed and predicted data with respect to: (i) the relative frequencies of monomorphic F+, monomorphic F-, and bimorphic F+/F- bursts, respectively; (ii) the size distributions among F+, F- and F+/F- bursts; and (iii) the proportions of subclones expressing Hb F within bimorphic F+/F- bursts. Given the hypothesis that erythroid progenitors which have an active Hb F program are less differentiated than cells which do not proceed to express Hb F, the stochastic event indicated by our analyses may be the probability that adult erythroid progenitor cells undergo terminal differentiation at an earlier stage than usual.

Low-dose actinomycin D (Acto) selectively suppresses murine erythropoiesis without decreasing erythropoietin (Ep) production. We used the plasma clot system to determine the stage of erythroid differentiation at which this inhibition occurs. Late erythroid precursors, CFU-E, and less differentiated committed erythroid stem cells, BFU-E, were assayed in CF1 mice given Acto 75–82 microgram/kg/day or saline subcutaneously for 5 days. We also assayed pluripotent (CFU-S) and committed granulocyte-monocyte (CFU-C) stem cells. Reticulocytes and marrow and spleen nucleated erythroid precursors were decreased by 99% in the Acto-treated mice; tibial marrow CFU-E were decreased by 97% and splenic CFU-E by 99%. Tibial BFU- E were not decreased by Acto, although there was a 66% diminution in splenic BFU-E. Acto increased tibial CFU-S, but splenic CFU-S and tibial and splenic CFU-C were unchanged. Thus Acto inhibits erythropoiesis by suppressing the ability of immediate committed erythroid precursors of CFU-E or CFU-E themselves to differentiate further in response to Ep. Acto does not affect survival or proliferation of the less differentiated cells--CFU-C, CFU-S, and marrow BFU-E. The suppression of splenic BFU-E in Acto-treated mice may indicate that marrow and splenic BFU-E are basically different stem cells. Alternatively, Acto treatment may impair migration of BFU-E from marrow to spleen.

Changes in haematopoiesis which occur in humans after burning injury may have important effects on morbidity and mortality. Because of the heterogeneity of burn patients we studied the regulation of blood cell formation which occurs in an animal using an established mouse model. Mice received a 20 per cent third degree scald injury on the back. Serial studies of a variety of haematopoietic parameters including stem cell, bone marrow and peripheral blood findings were done post burn. Although anaemia occurred frequently after injury red blood cell survival studies and examination of the stool for occult blood showed that neither haemolysis nor blood loss were primary causes of the anaemia. Bone marrow erythroid stem cells fell markedly post burn and this was associated with the development of a substance in serum capable of inhibiting red cell colony formation but not white cell colony formation of normal marrow cells. Reticulocytosis occured but was mild and the anaemia was primarily of the aregenerative type. Partial compensation for the depressed marrow erythropoiesis occurred in the spleen with an increase in erythroid colony-forming cells and erythroblasts. Marked granulocytosis occurred in the peripheral blood and bone marrow. There was an increase in splenic granulocytic stem cells post burn. Megakaryocytosis was striking in the bone marrow and spleen and there was an increase in peripheral blood platelet count. Evidence of immune stimulation included an increase in the size of the spleen and an increase in peripheral blood and splenic lymphocytes. Correlations of many of these findings suggested that the events were not occurring at random but that the changes in haematopoiesis were linked together. We speculate that the anaemia was the result of the increase in granulopoietic and thrombopoietic effort seen post burn.

Ouabain enhances the number of clonally derived erythroid stem cell colonies (CFU-e and BFU-e) from normal murine bone marrow. Ouabain is known to inhibit lymphocyte proliferation by blocking the Na+/K+ pump. To further explore these Na+/K+ ATPase-associated interrelationships, valinomycin, an inhibitor of Na+/K+ ATPase was employed. The addition of valinomycin (10-11 to 10-15M) in the presence of ouabain and erythropoietin (Ep) did not alter the erythroid colony-forming stimulation which was characteristic of cultures in which only ouabain (10-15M) and Ep were added. Valinomycin did effectively block both CFU-e and BFU-e formation when it was added in the range of 10-3 to 10-9M. The mechanism of this valinomycin-induced inhibition and ouabain stimulation is discussed.

To determine the role of natural killer (NK) cells in the regulation of human erythropoiesis, we studied the effects of NK-enriched cell populations on the in vitro proliferation of erythroid stem cells at three different levels of maturation (day 14 blood BFU-E, day 5–6 marrow CFU-E, and day 10–12 marrow BFU-E). NK cells were enriched from blood by Percoll density gradient centrifugation and by fluorescence- activated cell sorting (FACS), using the human natural killer cell monoclonal antibody, HNK-1. The isolated enriched fractions were cocultured with autologous nonadherent marrow cells or blood null cells and erythropoietin in a methylcellulose erythroid culture system. Cells from low-density Percoll fractions (NK-enriched cells) were predominantly large granular lymphocytes with cytotoxic activity against K562 targets 6–10-fold greater than cells obtained from high- density Percoll fractions (NK-depleted cells). In coculture with marrow nonadherent cells (NA) at NK:NA ratios of 2:1, NK-enriched cells suppressed day 5–6 CFU-E to 62% (p less than 0.025) of controls, whereas NK-depleted cells slightly augmented CFU-E to 130% of controls (p greater than 0.05). In contrast, no suppression of day 10–12 marrow BFU-E was observed employing NK-enriched cells. The NK CFU-E suppressor effects were abolished by complement-mediated lysis of NK-enriched cells with the natural killer cell antibody, HNK-1. Highly purified HNK- 1+ cells separated by FACS suppressed marrow CFU-E to 34% (p less than 0.025) and marrow BFU-E to 41% (p less than 0.025) of controls. HNK- cells had no significant effect on either BFU-E or CFU-E growth. NK- enriched cells were poor stimulators of day 14 blood BFU-E in comparison to equal numbers of NK-depleted cells or T cells isolated by E-rosetting (p less than 0.01). Interferon boosting of NK-enriched cells abolished their suboptimal burst-promoting effects and augmented their CFU-E suppressor effects. These studies provide evidence for a potential regulatory role of NK cells in erythropoiesis. The NK suppressor effect is maximal at the level of the mature erythroid stem cell CFU-E. These findings may explain some hypoproliferative anemias that develop in certain NK cell-activated states.

Patients with type I diabetes mellitus treated with continuous sc insulin infusion (CSII) have improved glucose homeostasis, metabolic control, and linear growth. To determine the influence of CSII on cellular growth in vitro, we used a clonal stem cell assay for proliferation of erythroid progenitors, [burstforming units-erythroid (BFU-E)] in peripheral blood. Eight patients were studied before and after 1 week of CSII. Improvement in metabolic control was demonstrated by a decrease in mean 24-h plasma glucose from 232 +/- 29 (+/- SEM) mg/dl before treatment to 112 +/- 3 mg/dl after treatment (P = 0.01). Somatomedin-C levels increased from 1.1 +/- 0.4 to 1.4 +/- 0.4 U/ml (P less than 0.001). Numbers of BFU-E-derived colonies were not different from normal during conventional treatment, but increased 300% after 1 week of CSII. Our findings indicate that the acute metabolic and hormonal improvements that accompany short term CSII therapy in vivo are associated with a striking increase in the proliferation of erythroid committed stem cells in vitro.

The synthesis of γ- and β-globin chains was investigated in erythroid cultures from first and second trimester fetuses as well as neonates in order to examine whether the differences in production of HbA and F in the fetal and newborn ontogenetic stages reflect in vivo predetermined differences in commitment among erythroid progenitors. Cultures of erythroid progenitors from first and second trimester fetuses (12 experiments) produced amounts of HbF that were similar to those synthesized in vivo by fetal liver erythro-blasts and reticulocytes (range of HbF production: in vivo, 84%-93%; in culture, 80%-94%). There was no difference in HbF synthesis in cultures of fetal liver or peripheral blood-origin progenitors from the same fetus. In cultures of circulating progenitors from full-term newborns (nine experiments), amounts of fetal hemoglobin that are expected for the newborn ontogenetic stage (range of HbF in culture: 55%-80% of total Hb) and that are close to the levels of HbF production in cord-blood reticulocytes of the same fetuses, were observed. Varying the concentration of erythropoietin by 40-fold produced only minor changes in the proportion of γ and β chain synthesis in culture. The findings show that the fetal or newborn erythroid stem cells are programmed in vivo for expressing the characteristic, for each ontogenetic stage, pattern of non-α-globin synthesis. This is consistent with the proposition that the regulation of γ- and β-chain synthesis during development takes place at the level of erythroid progenitor cells rather than in the terminally mature erythron.

In order to characterize the target cell for the polycythemia inducing Friend virus (FV-P) in vivo, mice were treated by induction of plethorism, bleeding, Actinomycin D, and Busulfan before virus infection. The development of the Friend leukemia was then studied mainly using the CFUE technique for erythroid colony growth in vitro. This technique allows the quantification of a new cell type, an erythropoietin (Ep) independent colony forming cell. These Ep independent colonies were taken as marker for the disease. Their number with time after infection was correlated with the compartment size of pluripotent, granuloid committed and erythroid stem cells at the time of infection. The results indicate that the development of the Friend leukemia does not require the actual presence of CFUE, as seen using Actinomycin D, and is not correlated with the number of pluripotent or granuloid stem cells, as seen after Busulfan. It is, however, dependent on the erythropoietic state of the animal, as seen in plethoric mice and mice after bleeding. It is, therefore, concluded that the target cell for FV-P is located within the Ep-responsive cell compartment, between early (BFUE) and late (CFUE) erythroid precursor cells.

A patient heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase and with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) was treated with combination chemotherapy and had a partial loss of Philadelphia chromosome accompanied by partial restoration of nonclonal hematopoiesis as determined by glucose-6-phosphate dehydrogenase. Studies of in vitro hematopoiesis were performed after chemotherapy to evaluate the influences of neoplastic stem cells on normal cells and to determine whether there were physical and cell kinetic differences between leukemic stem cells and their normal counterparts. The data revealed the following: (a) The frequencies of normal committed granulocytic stem cells (CFU-C) and erythroid stem cells (BFU-E) in blood did not differ from the frequencies in marrow. (b) Normal late erythroid progenitors (CFU-E) were found at a significantly lower frequency that the more primitive BFU-E. Calculations indicated that not only was there a decrease in CFU-E production by normal BFU-E, but there was also abnormal clonal expansion of CML BFU-E (CFU-E:BFU-E ratio for normal progenitors was 1.1, whereas for the CML clone it was 11.5). (c) No increase in frequency of normal CFU-C was found after marrow cells were exposed to high specific activity tritiated thymidine. (d) Normal CFU-C and those from the CML clone were not separable on the basis of density. (e) The frequency of normal BFU-E was consistently greater than that of CFU-C, suggesting that regulatory differences influence the commitment of normal progenitors to the two pathways.

Various culture systems are now available to study erythropoiesis in vitro. These include suspension and cloning culture techniques. Suspension culture techniques are frequently used for studying biochemical changes in erythroid cells, while cloning culture techniques are suited for investigating the nature of erythroid stem cells. Some aspects of human erythropoiesis will be clarified by employing new methods such as the Marbrook diffusion chamber technique.

Recent developments have focused on long-term bone marrow cultures and serum-free media to establish clonal hematopoiesis in vitro. Prior investigations have suggested an important role for such ingredients as transferrin, albumin, lipid, selenite, and potassium provided by serum. These factors, when present, can support clonal growth even in the absence of serum. Results reported here further define the importance of the cations K+ and Li+ in the regulation of in vitro erythropoiesis in the presence of both normal and dialyzed fetal calf serum and ouabain. While K+ proved essential for optimal erythroid colony formation, the presence of Li+ in such cultures reduced optimal colony formation of erythroid stem cells. This evidence suggests that in long-term marrow cultures or serum-free media K+ is essential for successful maintenance of self-renewal of erythroid stem cells and their differentiation, and that the monitoring of K+ levels may prove useful in such cultures. Since in vitro erythropoiesis was reduced in the presence of Li+, the mechanisms controlling differentiation of hematopoietic stem cell may be interpreted to be subject to cation influence.

Four children with congenital hypoplastic anemia (Diamond-Blackfan syndrome) and 30 control children with normal erythropoiesis were studied by a cell culture method in which human marrow, grown in a plasma clot, responds to added erythropoietin (EPO) with the appearance of discrete colonies of nucleated erythroid cells. The colonies arise from EPO-responsive stem cells and are not related to the number of morphologically identifiable marrow erythroids plated. Results of studies on control marrow indicated that without EPO there was little or no colony formation. Increasing EPO doses or nucleated marrow cells per culture resulted in a linear increase in colony numbers. The optimal EPO concentration of 2.5 U/ml yielded a mean of 158 +/- 79 colonies/1 x 10(5) nucleated cells on day 7 of incubation. Even in the absence of recognizable erythroids, marrows of all four patients with anemia grew erythroid colonies. Two patients on no therapy had decreased colony numbers. The other two, on prednisone, had normal numbers. Sera from patients did not inhibit colony formation from either autologous or control marrow. In contrast, serum from an adult with acquired pure red cell aplasia produced striking inhibition of colony growth. It appears that the red cell failure in this disorder is not due to an absence of erythroid stem cells, and a serum inhibitor to erythropoiesis as seen in the acquired disease is unlikely.

Serial microscopic immunodiffusion assays of F cells, i.e., erythrocytes that contain fetal hemoglobin (HbF), in four individuals recovering from anemia demonstrate initial increases in the percentage of circulating reticulocytes that contain HbF (F reticulocytes) and subsequent increases in the percentage of mature erythrocytes that contain HbF (F erythrocytes). In one individual responding to therapy for iron-deficiency anemia, the average percentage of F reticulocytes increased from 4.8+/-1.1 to 16.0+/-2.8% (mean+/-SD), while the mean level of F erythrocytes increased from 3.5+/-0.7 to 7.2+/-0.6%. Two normal children with transient erythroblastopenia exhibited F reticulocyte percentages of 71.3+/-6.7 and 41.5+/-1.5%, respectively, when erythropoiesis resumed. With recovery these values fell to finally measured values of 33.7+/-4.7 and 12.6+/-1.1%, respectively. In an adolescent with sickle cell anemia, F-reticulocyte percentages fluctuated between 0.6+/-1.1 and 34.0+/-2.8% and paralleled the rise and fall of total reticulocytes associated with therapy for a nasopharyngeal carcinoma. Such findings suggest that first, the production of F cells and non-F cells are separately regulated. Second, F-cell production is preferentially stimulated during escape from erythropoietic suppression and selectively depressed at the start of suppression. Third, during escape from erythropoietic suppression, F-cell production in vivo resembles that reported for in vitro cultures of erythroid stem cells. Fourth, individuals with sickle cell anemia, like individuals without hemoglobinopathies, can change their relative level of F-cell production.

Hemoglobin switching and macrocytosis were studied in homozygous hemoglobinAA sheep. An abrupt initiation of erythropoietic stress, accompanied by a pulsed elevation of circulating erythropoietin levels, was induced by phlebotomy. Sequential blood samples were separated according to density on Stractan gradients to isolate cells newly entering the circulation from the marrow each day. Analysis of hemoglobin phenotype and cell volume distribution in these young reticulocytes revealed a distinct temporal separation in the appearance of hemoglobin C and increased cell volume. The appearance of macrocytes within 24 hr of erythropoietin elevation suggests that macrocytosis could be the result of the action of erythropoietin during the late stages of erythroid maturation. The 72-hr delay in the appearance of hemoglobin C indicates that commitment to a particular hemoglobin phenotype occurs at an early stage of differentiation and involves immature erythroid stem cells. The results of this study show that these consequences of erythropoietic stress are initiated at two different developmental stages, resulting in the production of macrocytosis and hemoglobin switching.

Assay of red cell progenitors by colony formation in culture is expected to allow study of early events in erythroid differentiation in animal models and in man. In this communication, a simplification of the culture method for mouse bone marrow cells originally reported by Stephenson et al. ('71) is described. In the modified procedure, plasma clot is replaced by methyl cellulose, and scoring of erythroid colonies is done directly in the plates without staining. With additional modification the system proved applicable to the culture of erythroid colonies from human bone marrow as well. The development of both mouse and human erythroid colonies was dependent on “erythroid colony stimulating activity” (E-CSA) supplied by extracts of anemic sheep plasma, or by extracts of urine from anemic patients. Over a certain range, the number of colonies was a function of dose of E-CSA. In addition to E-CSA, these extracts also possessed erythropoietin activity as measured in plethoric mice. The possible chemical equivalence of urinary E-CSA and erythropoietin was suggested by their similar behavior on both gel filtration and affinity chromatography on agarose-concanavalin A. The finding further suggests that the culture method might prove useful for the bioassay of erythropoietin. Granulocyte colony stimulating activity (G-CSA) was also present in the urine extracts, as detected in cultures of mouse bone marrow. Virtually all of this activity was bound on agarose-con A, whereas only a small fraction of E-CSA was retained on this material. Agarose-con A may thus be useful for the purification of erythropoietin.

In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

We have found that the peripheral blood of normal humans contains a significant number of committed erythroid stem cells of high proliferative capacity. These erythroid stem cells closely resemble the murine erythroid burst-forming unit (BFU-E) with respect to proliferative capacity, colony morphology, and erythropoietin requirement. BFU-E were isolated from the peripheral blood of normal individuals by Ficoll-Hypaque density gradient centrifugation and cultured in vitro using the plasma culture technique. Macroscopic erythroid colonies of between 100 and 1000 cells were observed after 10-14 days of culture in the presence of either sheep or human erythropoietin at 0.5-4 units/ml. Individual colonies contained between 3 and 20 subcolonies and reached a maximum mean size of approximately 500 cells. Colony number was linearly related to the cell input, suggesting that a single cellular entity was precursor to each colony. The frequency of cells capable of giving rise to an erythroid colony was at least 100 per ml of blood in a number of individuals tested. The ability to assay significant numbers of erythroid precursor cells of high proliferative capacity from normal peripheral blood should facilitate the study of both normal erythropoiesis and of disease states affecting erythropoiesis in which marrow samples are not available on a routine basis.

To investigate whether the level of maturity of human erythroid cells influences the expression of the fetal hemoglobin program, we studied the relative production of fetal (Hb F) and adult (Hb A) hemoglobins during the maturation of erythroid clones produced in vitro by adult or neonatal erythroid stem cells. In both the adult and the neonatal cell cultures, clones composed of immature erythroblasts showed a significantly higher Hb F/Hb A ratio compared to the mature clones. Culture conditions enhancing erythroid cell maturity (such as an increase in the level of erythropoietin or culture time) decreased the relative synthesis of Hb F in the maturing erythroid cells. Direct immunofluorescence studies demonstrated earlier production of Hb F compared with Hb A during maturation of adult-origin HbF-synthesizing clones. The findings show that the final expression of Hb F is influenced by the degree of maturity of the terminally differentiated cells and suggest that, in addition to regulation at the level of erythroid stem cells, there is control of Hb F expression during erythroblast maturation. The inverse relationship between Hb F expression and level of cell maturity suggests that a regulatory mechanism operating throughout the process of erythroid stem cell differentiation/erythroblast maturation decreases the potential of Hb F expression as the development of the erythroid cell advances.

Two types of erythroid colonies were generated in vitro from sheep fetal liver cells. The first type consisted of single colonies of 8–256 cells that were well hemoglobinized by 4 days; these are thought to originate from CFU-E. The second type consisted of macroscopic colonies composed of several subcolonies that matured between days 3 and 8 in vitro. At maturity, each contained 256 to > 1000 cells that formed a discrete macroscopic cluster. The macroscopic colonies, not previously described in sheep, are thought to be derived from BFU-E. The characteristics of sheep BFU-E were defined and the production of fetal hemoglobin (HbF, alpha 1, gamma 2) and HbC (alpha 2 beta 2) was compared in colonies derived from CFU-E or BFU-E. Bursts developed at erythropoietin (epo) concentrations as low as 0.1 U/ml, although the number observed increased with epo concentration up to 10 U/ml. The number of bursts observed was approximately proportional to the number of cells plated. As shown by thymidine suicide, approximately 50% of both the BFU e and CFU-E were in S-phase when obtained from the fetus. BFU-E were smaller and partially separable from CFU-E after sedimentation at unit gravity. The beta c/gamma synthetic ratio in colonies derived from BFU-E was greater than in CFU-E-derived colonies. These data suggest that the capacity for generation of erythroblasts making HbC is greater in the earlier or more primitive erythroid stem cells in fetal liver.

An impaired erythropoietic response to anemia has been noted in human patients with malaria and in rodents experimentally infected with Plasmodium berghei. We have attempted to characterize the erythropoietic response in mice with a fatal P berghei infection, with particular emphasis on changes in marrow hematopoietic stem cells. Mice infected with P berghei had dramatic decreases in bone marrow cellularity, erythroblasts, BFU-E, and CFU-E as early as 24 hours postinfection and before there was any change in hematocrit. With development of anemia, marrows became erythropoietic with some expansion of the CFU-E compartment, but the BFU-E pool remained depleted and reticulocyte response was inadequate. There was no significant change in CFU-S from marrows of malaria-infected mice one day after infection. The lethality of malaria infection may take three weeks to be revealed, but it may be determined within hours of the infection by the irreparable changes in marrow erythroid stem cells.

The requirement of erythropoiesis for erythropoietin were studied in a patient with Ph chronic myelocytic leukemia who had undergone an erythroblastic transformation. Transfusions resulted in a suppression of erythropoiesis. Plasma clot culture studies indicated that both the CFU-E and BFU-E in the peripheral blood of this patient were dependent upon erythropoietin for their differentiation and proliferation. Neither of these committed erythroid stem cells was cloned in the absence of erythropoietin. These studies suggest that the proliferation and differentiation of erythroid stem cells during the erythroblastic crisis of this disorder remain dependent upon physiologic regulators.

Results reported herein describe the expansion of erythroid stem cells (CFU-e and BFU-e) from marrow and spleen cells from mice infected with Rauscher leukemia virus (RLV). Maximum colony numbers were seen by 5 weeks after viral inoculation. The increase in splenic CFU-e and BFU-e was related to development of splenomegaly, the latter being a characteristic feature of murine oncornavirus infection. Both splenic and marrow RLV-derived CFU-e were erythropoietin (Ep) dependent. However, splenic and marrow RLV-derived BFU-e proliferation occurred at Ep levels considered suboptimal for normal colony formation. This increased erythroid stem cell proliferation may be due in part to the increased demand on erythropoiesis as a result of the profound fatal anemia which occurs in RLV-induced erythroleukemia. These studies suggest that factors operating at the level of BFU-e may enhance erythropoiesis, and hence may be a direct result of murine oncornavirus infection.

Plethoric mice treated with pharmacological doses of estradiol have decreased concentration of erythropoietin-responsive cells (ERC) in the marrow. We used the methylcellulose-culture system for growth of erythroid stem cells (CFU-E and BFU-E) to define more accurately these estrogen-induced changes. As an animal model we utilized plethoric mice given repeated injections of estradiol cypionate and found that at 14 days after the onset of treatment there was no significant change in the concentration of femoral CFU-E whereas there was a significant decrease of the BFU-E content. Both CFU-E and BFU-E increased progressively in the spleen over a 42-day period. Addition of estradiol directly to the cell-culture system showed no effect on CFU-E growth but induced a significant depression of BFU-E growth. This depression seemed to require the presence of adherent cells. It is our hypothesis that estrogens suppress only the early stages of erythroid proliferation and/or differentiation by a mechanism involving possibly the stromal (adherent) cells of the marrow microenvironment.

Human mononuclear leukocytes were fractionated into populations of null, T and B cells by immunoabsorbent column chromatography followed by E-rosette formation and purification of T cells by differential centrifugation and osmotic lysis. The unfractionated and fractionated cell populations were first separately cultured for 14 days in plasma clots in the presence of two international units erythropoietin. Typical erythroid burst-forming unit (BFU-E)-derived colonies grew in the unfractionated cell cultures but not from T- or B-cell cultures. BFU-E colonies grew in null cell cultures but most of the colonies were small and variably hemoglobinized with less than three subcolonies. When intact T cells were added to null cells and cocultured, many typical large BFU-E colonies with more than 10 well homogenized subcolonies appeared. Increasing numbers of large BFU-E colonies in null cell cultures were induced by stepwise addition of T cells but not by the addition of B cells. A conditioned medium in which T cells had been induced to divide by tetanus toxoid substituted for intact T cells in this T-cell-dependent BFU-E colony formation observed in null cells. These findings demonstrate that the BFU-E, a committeded erythroid stem cell, resides in the null cell fraction of peripheral blood, but its proliferative capacity and differentiation in vitro requires a soluble product of T cells. Such experiments now permit a new approach to the assessment of various disorders of erythropoiesis. Erythroid hypoplasia in a particular case may be due to dysfunction of the committed precursor cell or to a failure of a helper effect induced by T cells.

Using thiamphenicol and scheduled bleeding, we were able to induce an adequate number of erythroid stem cells (CFU-e) in mice in order to conduct an in vivo study of the changing expression of c-myc and c-fos oncogenes during erythropoiesis. Results indicated that c-myc and c-fos are active in erythropoiesis and have a similar pattern of expression. A large decrease in expression of both c-myc and c-fos occurs when erythroid cells begin the biochemical transition into mature phenotypes, i.e., when RNA synthesis is down-regulated.

Using the technique for erythroid colony formation in vitro, bone marrow and spleen cells from NMRI mice were studied after Rauscher virus (RLV) infection. There was a substantial decrease in CFUE concentrations during the first days after infection, this being more pronounced in the marrow than in the spleen. In the marrow values gradually return to and are maintained at control levels, while in the spleen a 40-50-fold increase is seen between days 8 and 18. In normal and RLV infected animals the same dose dependence of CFUE growth for erythropoietin was seen. For normal and RLV infected cells in the absence of erythropoietin there were only very few background colonies. In exhypoxic plethoric mice the increase in CFUE concentration seen in normal mice in the spleen, is delayed by 2-3 days.

A 5-year-old girl developed pure red cell aplasia (PRCA) during sodium valproate (VPA) administration. Only four cases of VPA induced PRCA have been reported in the literature. Furthermore no precise report concerning the underlying mechanisms is available. Using a system for the clonal growth of autologous bone marrow committed erythroid stem cells (CFU-E, BFU-E) VPA itself was not inhibitory for colony formation at a physiological concentration. Patient's serum and peripheral mononuclear cells collected at admission showed no inhibitory effects on erythroid progenitor cell growth. Rapid recovery from PRCA was observed after discontinuation of VPA, without immunosuppressive therapy. Our observation suggests that VPA may induce PRCA through the inhibitory effect beyond the differentiation stage of BFU-E and CFU-E in some cases.

A single injection of BCNU (LD-10 range) caused in mice a prolonged suppression of erythropoiesis, whereas granulopoiesis recovered within a few days. The suppression of erythropoiesis was due to a delayed restoration of erythropoietin-responsive cells (ERC), although the CFU were only moderately suppressed and ranged from 30% to 60% of normal. Daily erythropoietin injections promptly restored normal ERC populations without affecting CFU. Injections of androgens after BCNU had no effect on ERC restoration, but they markedly enhanced CFU regeneration and caused neutrophil counts up to ten times normal.

Five clonal human bone marrow stromal cell lines were isolated from the adherent cell populations in long-term liquid cultures after transfection with the recombinant plasmid pSV3gpt. All the cell-line feeder layers and their conditioned media stimulated the proliferation of committed granulomonocytic stem cells (CFUc) from human bone marrow. The size and number of early erythroid stem cell (BFUe)-derived colonies were significantly increased when in the presence of 10% conditioned medium from the cell lines. Furthermore, a substantial number of mixed colonies with erythroid components were observed in the cultures in the presence of erythropoietin and conditioned medium. These findings suggest that the human bone marrow stromal cell lines obtained after transfection with pSV3gpt may be extremely useful in identifying the hematopoietic factors derived from the hematopoietic microenvironment and in analyzing their mechanism of action.