Purification of human blood burst-forming units-erythroid and demonstration of the evolution of erythropoietin receptors

To facilitate the direct study of the molecular events that control the development of human burst-forming units-erythroid (BFU-E), we have developed a method to purify BFU-E from peripheral blood. Using density centrifugation, rosetting with a mixture of neuraminidase-treated and IgG-coated sheep erythrocytes, positive panning with anti-My10 monoclonal antibody, overnight adherence to plastic dishes, negative panning with monoclonal antibodies, and density centrifugation, human blood BFU-E were purified from 0.04% to 56.6%, a 1,400-fold purification with a 13% yield. More than 90% of purified BFU-E were recombinant interleukin-3 (rIL-3) dependent, which survived for 48 h with rIL-3 in the absence of recombinant erythropoietin (rEP), and 80% gave rise to erythroid bursts of more than 500 hemoglobinized cells. rEP dependency was not evident until after 72 h of incubation in vitro. The purified cells (day 1) were incubated with rIL-3 and rEP in liquid culture for 24 (day 2), 48 (day 3), and 72 (day 4) h and then were transferred into semisolid cultures and incubated until day 15. The size of the erythroid colonies observed in semisolid cultures decreased continuously in association with the incubation time of day 1 purified cells in liquid cultures. The first appearance of colony-forming units-erythroid (CFU-E) that gave rise to colonies of 8 to 49 cells was observed after 72 h of incubation of day 1 cells in the liquid culture. 125I-rEP was incubated for 5 h at 37 degrees C with purified cells (day 1) or with the cells that had been incubated in liquid culture for an additional 24-72 h, and the presence of erythropoietin (EP) receptors was investigated using autoradiography. Specific binding of 125I-rEP was detected in 19 +/- 7% of the initial day 1 BFU-E. The percentage of 125I-rEP-binding to erythroid progenitor cells and the amount of binding continuously increased as day 1 BFU-E matured. 125I-rEP specific binding was observed with all of the erythroid progenitor cells that had been incubated in liquid culture for 72 h. These data demonstrate that primitive BFU-E have a much lower number of EP receptors than CFU-E and develop an increased concentration of EP receptors in association with their maturation and loss of proliferative capacity.     

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.     

Effects of androgens on burst forming units (BFU-E) in normal rabbit bone marrows

The mechanism of action of androgenic steroids on erythropoiesis is not well understood. In order to assess whether the site of action of androgens is on the early erythroid committed stem cell compartment, the $\text{in}$$\text{vitro}$ effects of testosterone (T), 5α-dihydrotestosterone (5α-DHT) and 5β-dihydrotestosterone (5β-DHT) on the so-called erythropoietic burst forming unit (BFU-E) in normal rabbit bone marrows were studied. Even though all of the steroids studied increased the number of BFU-E in the presece of Ep, 5β-DHT was the most potent in stimulating BFU-E. Testosterone was moderately effective in increasing BFU-E. Even though 5α-DHT produced a significant increase in BFU-E, it was the least effective of the 3 steroids studied. Preincubation (2 hrs) of normal rabbit bone marrow cells with testosterone followed by removal of T from the culture system resulted in a significant increase in BFU-E when compared with that of non-treated marrow cells in the presence of Ep. These data suggest that testosterone and 5β-DHT and possibly 5α-DHT act on an early uncommitted stem cell, perhaps the CFU-S, to increase the numbers of erythroid committed stem cells to eventually cause an increase in erythropoiesis in combination with Ep.     

Biosynthesis of globin chains in fetal liver and adult marrow cultures : Comparative analysis of individual colonies derived from early, intermediate or late erythroid progenitors

The relative synthesis of globin chains (α,β,^Gγ,^Aγ) has been comparatively evaluated in erythroid colonies from 26 fetal livers (7–15 gestational week) and 13 ‘normal’ adult marrows. Clusters deriving from erythroid colony-forming units (CFU-E) were analysed either individually or in pools of –20 colonies. Bursts deriving from earlier erythroid progenitors (erythroid burst-forming unit, ‘primitive’ or ‘mature’, P-BFU-E or M-BFU-E, respectively) were always analysed individually. Since γ-globin synthesis peaks earlier than β-chain production in both the fetal and the adult erythroblastic pathway, the globin synthetic pattern has been comparatively evaluated, in so far as possible, in colonies at an homogenous, advanced stage of hemoglobinization.In fetal liver cultures, the relative β-synthesis in CFU-E clusters, M- and P-BFU-E bursts constantly shows low, fairly uniform values. In adult marrow cultures, the relative γ-production in the corresponding three classes of colonies is characterized by low, rather homogeneous levels (except for more elevated γ-synthetic values occasionally observed in pooled CFU-E clusters comprising a majority of poorly-hemoglobinized colonies). A gradual decrease of relative γ-production has never been observed in colonies deriving from progressively more differentiated erythroid progenitors of both fetal and adult origin.These results suggest that fetal and adult BFU-E are endowed respectively with a program for prevailing HbF or HbA synthesis, which is not substantially modulated at the level of erythroid progenitors under standard culture conditions. By implication, it is postulated that, in fetal and more particularly adult age, modulation of globin synthesis is mediated via mechanism(s) acting at the level of erythroblasts, i.e. at the level of the early γ- and the late β-synthesis in their maturation pathway. The Hb switch (i.e. the switch from prevailingly HbF to HbA synthesis program) is possibly dependent on the ontogenic ‘maturation’ of BFU-E (and/or stem cells), which peaks in the perinatal period.     

THE ROLE OF ERYTHROPOIETIN IN REGULATION OF POPULATION SIZE AND CELL CYCLING OF EARLY AND LATE ERYTHROID PRECURSORS IN MOUSE BONE MARROW

This study was designed to determine the stage in haemopoietic cell differentiation from multipotential stem cells at which erythropoietin becomes physiologically important. The responses of haemopoietic precursor cells were monitored in the bone marrow of mice under conditions of high (after bleeding) and low (after hypertransfusion) ambient erythropoietin levels. The number of relatively mature erythroid precursors (CFU-E), detected by erythroid colony formation after 2 days of culture, increased three-fold in marrow by the fourth day after bleeding, and decreased three-fold after hypertransfusion. Assessed by sensitivity to killing by a brief exposure to tritiated thymidine (³H-TdR) in vitro, the proliferative activity of CFU-E was high (75% kill) in untreated and bled animals, and was slightly lower (60% kill) after hypertransfusion. The responses of more primitive erythroid progenitors (BFU-E), detected by erythroid colony formation after 10 days in culture, presented a contrasting pattern. After hypertransfusion they increased slightly, while little change was noted until the fourth day after bleeding, when they decreased in the marrow. The same response pattern was observed for the progenitors (CFU-C) detected by granulocyte/macrophage colony formation in culture. The sensitivity of BFU-E to ³H-TdR was normally 30%, and neither increased after bleeding nor decreased after hypertransfusion. However, in regenerating marrow the ³H-TdR sensitivity of BFU-E increased to 63%, and this increase was not affected by hypertransfusion. These results are interpreted as indicating (1) that physiological levels of erythropoietin do not influence the decision by multipotential haemopoietic stem cells to differentiate along the erythroid pathway as opposed to the granulocyte/macrophage pathway; (2) that early erythroid-committed progenitors themselves do not respond to these levels of erythropoietin, but rather are subject to regulation by erythropoietin-independent mechanisms; and (3) that physiological regulation by erythropoietin commences in cells at a stage of maturation intermediate between BFU-E and CFU-E.     

Acetylation of human fetal hemoglobin occurs throughout erythroid cell maturation

The biosynthesis of human acetylated fetal hemoglobin (Hb F1) has been examined by incubating the following cell types with [3H]leucine: (a) burst-forming unit erythroid cells cultured from umbilical cord mononuclear cells, (b) infant bone marrow, (c) umbilical cord blood, and (d) peripheral blood cells from adults with elevated fetal hemoglobin. Newly synthesized Hb F1 was 18-20% that of Hb F0 in burst-forming unit erythroid cells which were immature, mature, or in an intermediate state of development. In infant marrow and in infant and adult peripheral blood the extant Hb F1 comprised 10.8 +/- 1.8% of the total Hb F. In marrow cells the specific radioactivity (cpm/mg) of Hb F1 was 1.4-2.0-times greater than that of Hb F0. In peripheral blood cells these ratios were slightly greater. [3H]Leucine-labeled infant bone marrow, umbilical cord blood, and adult peripheral blood cells were subjected to density gradient ultracentrifugation. The ratios of specific radioactivity for Hb F1/Hb F0 increased from 1.0-1.8 in the lightest cell zone to 5.2-9.0 in the more dense cells. Thus the biosynthesis of Hb F1 is enhanced in cells which are more mature than those responsible for the bulk of hemoglobin synthesis, and the acetylation of Hb F provides a marker of erythroid cell maturation.     

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

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

Biosynthesis of Hb in individual fetal liver bursts : γ-Chain production peaks earlier than β-chain in the erythropoietic pathway

The relative synthesis of α-, β-, ^Gγ- and ^Aγ-globin chains has been evaluated in single fetal liver bursts, which were grown in methylcellulose cultures, individually labelled with [³H]leucine and then analysed via iso-electric focusing. Well-hemoglobinized bursts demonstrate a homogeneous globin synthetic pattern, characterized by prevalent HbF (+some HbA) synthesis: thus, they apparently originate from a homogeneously programmed population of erythroid burst-forming unit (BFU-E). On day 8–9 of culture, the synthetic pattern in ‘mature’ (i.e., well-hemoglobinized) bursts has been compared with that in simultaneously-grown, ‘immature’ (i.e., poorly-hemoglobinized) colonies. These patterns have been further compared with that in ‘matured’ bursts (identified in situ as immature on day 8–9 and labelled 2–4 days later when matured). The ‘immature’ colonies showed very low levels of relative β-globin synthesis, while the ‘mature’ ones demonstrated a more elevated production of β-chain. Significantly, the ‘matured’ bursts showed a globin chain synthetic pattern similar to that of previously labelled ‘matured’ colonies. It is postulated therefore that in fetal liver (and also in adult marrow) the synthesis of γ-chain is linked to an early differentiation stage of erythroblasts, while β-globin synthesis is largely activated at a more advanced maturation stage.     

Stem cell factor can overcome inhibition of highly purified human burst-forming units-erythroid by interferon γ

Highly purified human blood burst-forming units-erythroid (BFU-E) were used to study the effects of interferon γ (IFNγ). IFNγ inhibited erythroid colony formation, cell proliferation, and differentiation of day 3 to day 6 mature BFU-E in a dose-dependent manner. The primitive BFU-E (day 1 and day 2 cells) and later day 7 cells were less affected. IFNγ dose-response experiments demonstrated that the number and size of erythroid colonies were reduced at a concentration of 500 U/ml with more complete inhibition at 1,000 U/ml. Inhibition of day 4 to day 6 erythroid progenitors was first noted by 72 h of incubation with IFNγ, and target cell growth and differentiation continued to decrease with further incubation. IFNγ also induced erythroblast apoptosis which was demonstrated by both nuclear condensation and fragmentation plus flow cytometry with in situ end-labelling. Because day 3 to day 6 cells need stem cell factor (SCF) for development in serum-free culture, the relationship of IFNγ inhibition to this growth factor was investigated. The reduction in the number of erythroid colonies by IFNγ was reversed by SCF although the colony size was not completely re-established. In contrast, interleukin-3 did not have the capacity to overcome the inhibitory effects of IFNγ. Since IFNγ blood levels are elevated in some anemias of chronic disease, IFNγ may have a role in promoting this anemia and its inhibitory effect might be better overcome by SCF plus EP. However, the mechanism by which these growth factors overcome the inhibition of IFNγ, or vice versa, is unknown at the present time. © 1995 Wiley-Liss, Inc.     

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.     

低氧对CD34^＋造血干／祖细胞增殖分化及其对细胞因子依赖性的影响

To elucidate the effects of hypoxia on the proliferation and differentiation of CD34(+) hematopoietic stem/progenitor cells and their response to cytokines, the cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Mononuclear cells (MNC) and CD34(+) cells were incubated in severe hypoxia (1% oxygen) culture system, and the colony forming cells and antigen expression were studied by colony forming assays and FACS analysis. The results showed that incubation in severe hypoxia increased the number of erythroid bursts (BFU-E) (324.8+/-41.4/10(4) cells) generated from CD34(+) cells (191.2+/-34.5/10(4) cells in the control group, P<0.01). Severe hypoxia also enhanced the maintenance and cloning efficiency of BFU-E in a liquid culture system without growth factors, the number of BFU-E (152.4+/-22.6/10(4)cells) was much bigger than that in the control group (74.2+/-9.3/10(4) cells, P<0.01). In cultures incubated in hypoxia, the percentage of CD34(+) cells was significantly higher (2.5+/-1.2-fold, P<0.05) than in those incubated in air. BFU-E cloning efficiency of MNC was not significantly affected by hypoxia. The above results show that hypoxia enhances the maintenance of erythroid progenitor cells generated from CD34(+) hematopoietic stem/progenitor cells, no matter growth factors are present or not. These positive effects of hypoxia did not occur for the other progenitors.     

Ex vivo expansion of primitive hematopoietic cells for cellular therapies: An overview

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34⁺ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood     

Testosterone action on BFU-E and CFU-E: correlation between enhancement of proliferative rate and amplification of pool size.

As previously reported, a single administration of testosterone propionate (TP) in ex-hypoxic polycythemic mice induces an 18–24 hr amplification of the erythroid burst-forming unit (BFU-E) pool and a 60-hr expansion of the erythroid colony-forming unit (CFU-E) compartment. Both phenomena are here shown to be temporally associated with an increase of the $\text{in vitro}$³H-TdR sensitivity of these compartments, thus indicating an elevation of their proliferative rate. On the other hand, no significant modification of both the DNA synthesis index and the pool size of BFU-E and CFU-E were observed at respectively 60 or 18 hr. At either time interval, both ³H-TdR sensitivity and compartment size were not modified at the level of the myeloid-macrophage colony-forming unit (CFU-C). It is therefore suggested that the early and late expansion of respectively BFU-E and CFU-E number after TP injection is at least partially mediated by enhancement of the proliferative rate within the respective compartments. Finally, mechanisms underlying TP action on BFU-E and CFU-E pools are discussed in the light of both present and previous observations.     

Haemopoiesis in the beagle foetus after in utero irradiation

On day 33 of gestation, foetal beagles were irradiated in utero (0.9 Gy of 60Co gamma-irradiation, 0.4 Gy/min). Foetal haematocytopoiesis was studied during the third trimester of gestation (days 42-55). Peripheral blood nucleated cell counts were 33 per cent lower than normal on day 44 and continued to be lower until day 49, when values became higher than normal. Splenic cellularities of irradiated pups on day 44 were more than 3 times those of the nonirradiated, but thereafter they were similar to normal. Differences in haemopoietic progenitor cell activity between irradiated and normal foetuses were observed. In comparison with the other foetal tissues, the foetal liver appeared to experience greater radiation injury. For example, on day 44, the irradiated liver BFU-E, CFU-E, and GM-CFC per 10(5) cells were almost fivefold lower than normal values. Spleens of irradiated foetal beagles contained a marked increase in all haemopoietic progenitor cells (BFU-E, CFU-E, and GM-CFC) and recognizable proliferative granulocytic cells and nucleated erythroid cells. The haemopoietic activity of the irradiated bone marrow during days 42-44 was similar to that of the irradiated spleen, and compensated for the damaged liver. However, unlike the irradiated spleen, the irradiated bone marrow had decreased BFU-E activity compared with the values for the nonirradiated bone marrow during days 48-55. Until day 50, the irradiated marrow contained fewer recognizable proliferative granulocytic cells but more nucleated erythroid cells.     

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.     

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

Summary A new method has been developed for the precise identification of human bone marrow colony forming unit erythroid (CFU-E) and burst forming unit erythroid (BFU-E) colonies, and for determination of the hemoglobin contents using microcytofluorometry. The method relies on a photochemical reaction in which intracellular hemoglobin is converted into fluorescent porphyrin under violet light (=405 nm) in the presence of an SH-donor (mercaptoethylamine hydrochloride). The CFU-E and BFU-E colonies showed red fluorescence with two spectrum peaks at 600 and 650 nm when illuminated by violet light. These two peaks are consistent with those of porphyrin fluorescence. The porphyrin fluorescence was not inducible in colony forming unit granulocyte-macrophage (CFU-GM) colonies, while 20% of the CFU-GM colonies were false positive with respect to the conventional benzidine reaction. The photochemically inducible fluorescence began to appear in BFU-E colonies on the 4th day of culture, while the same colonies started to be positive for the benzidine reaction on the 9th day. Therefore, the photochemical reaction was more specific and sensitive than the benzidine reaction for the identification of CFU-E and BFU-E colonies. In addition, this method enabled us to measure the hemoglobin level in the cells forming the colonies because the intensity of the fluorescence was proportional to the amount of hemoglobin when the photochemical reaction was carried out for 50 min. As a result of qualitative and quantitative analysis of CFU-E colonies by this method, it was possible to detect the hemoglobin levels in the colonies from 1 of 4 cases of untreated acute nonlymphocytic leukemia and from 2 of 4 cases of myelodysplastic syndrome in which the hemoglobin levels were too low to be detected by the benzidine reaction. These cases, where the CFU-E colonies showed very low levels of hemoglobin, were associated with poor prognosis. Thus, our method is useful for identifying CFU-E colonies, determining their hemoglobin synthesis, and as a cue to predict the clinical course of the patients.     

Inhibition of erythroid progenitor growth is mediated by cytotoxic lymphocytes and not by natural killer cells or IFN-gamma

Alloantigen primed T cells (PTC) were recovered from MLR at day 6 and 12, then added to cultures of erythroid progenitors, erythroid burst-forming units, BFU-E. The PBMC source of BFU-E was prepared either to retain or deplete APC by treatment with appropriate mAb and C. BFU-E grown in cocultures were counted at day 14 and replicate cultures assayed for IFN-gamma production on days 1 to 7. Analysis of MLR cells indicated that large, rapidly cycling cells recovered from MLR at day 6 have significant NK activity, whereas CTL activity is minimal, and production of IFN-gamma requires reexposure to APC. The smaller, noncycling cells recovered from MLR at day 12 have comparable NK activity, also require reexposure to APC for IFN-gamma production, but in addition have significant CTL activity. The addition of day 12 MLR cells to BFU-E cultures results in MHC restricted inhibition of BFU-E growth, suggesting that the CTL activity and not the NK activity contained within this population of cells is responsible for BFU-E inhibition. Studies using enriched population of BFU-E indicated that appropriate APC are needed to trigger both IFN-gamma production and BFU-E inhibition by the PTC. By using various APC-BFU-E combinations it was determined that after reexposure of PTC to appropriate APC, the inhibition of BFU-E was still target-specific indicating a direct effect between the PTC and BFU-E.     

Selection against blood cells deficient in hypoxanthine phosphoribosyltransferase (HPRT) in Lesch-Nyhan heterozygotes occurs at the level of multipotent stem cells

Lesch-Nyhan syndrome is caused by a severe genetic deficiency of hypoxanthine phosphoribosyltransferase (HPRT) and is characterized by central nervous system disorders, gout, and in some cases, macrocytic anemia. Women heterozygous for HPRT deficiency are healthy but their somatic cells are mosaic for enzyme deficiency owing to random inactivation of the X chromosome. Frequencies of red blood cells and T cells deficient in HPRT are significantly lower than the expected 50% in heterozygotes, suggesting that HPRT-negative blood cells are selected against in heterozygotes. To determine at which stage of hematopoiesis such selection occurs, we determined the frequencies of HPRT-negative T, B and erythroid precursor cells in three heterozygotes. Since the cloning efficiencies of T and B cells and colony forming efficiency of burst-forming unit erythroid (BFU-E) for sample from Lesch-Nyhan patients were similar to those of normal cells, HPRT deficiency does not seem to render the differentiated cells less efficient for proliferation. However, the frequencies of HPRT-negative T and B cells, and BFU-E were all less than 10% in each of the three heterozygotes. Although the frequencies of HPRT-negative cells showed tenfold variations between the heterozygotes, each heterozygote had similar frequencies of HPRT-negative cells in the three cell types. These results suggest that HPRT is important at early stages of hematopoiesis, but less so after the cells have differentiated into T cells, B cells and erythroid precursor cells.