Enhanced colony formation of human hemopoietic stem cells in reduced oxygen tension

In general, cell cultures, including hemopoietic stem cells, are produced in an atmosphere of various CO2 concentrations in air, although most cells in vivo proliferate and differentiate at lower oxygen tensions. We therefore investigated the effect of reduced oxygen tension on the in vitro colony growth of committed and multipotential hemopoietic progenitor cells from human bone marrow. All hemopoietic progenitor cells (CFU-mix, BFU-E, CFU-E, and CFU-GM) investigated showed enhanced colony growth at lower oxygen tension. CFU-E showed the highest enhancement, followed in order by BFU-E, CFU-mix and CFU-GM. At reduced oxygen tension, the sensitivity of early and late erythroid progenitor cells to erythropoietin was significantly increased, and this can be one of the mechanisms for the enhanced colony growth of erythroid progenitors. In the colony growth of CFU-GM, plating efficiency was also enhanced by the predominant increment of neutrophilic colonies. The lowering of oxygen tension would presumably reduce oxygen toxicity and result in the increased colony growth of human bone marrow stem cells, although the precise mechanisms of oxygen toxicity at the level of hemopoietic stem cells have yet to be elucidated. However, this clonal culture system, using a low oxygen tension, can be a useful means for elucidating the regulatory mechanisms involved in the proliferation and differentiation of hemopoietic progenitor cells in physiological and pathological conditions.     

Susceptibility of human erythropoietic cells to B19 parvovirus in vitro increases with differentiation

B19 human parvovirus is the etiologic agent of transient aplastic crisis. To better understand B19 virus-induced hematopoietic suppression, we studied the host cell range of the virus using in vitro bone marrow cultures. First, B19 virus replication was examined in the presence of various purified cytokines using DNA dot blot analysis. Replication was detected only in erythropoietin-containing cultures. The other cytokines (granulocyte/macrophage colony-stimulating factor [GM-CSF], G-CSF, M-CSF, interleukin-1 [IL-1], IL-2, IL-3, and IL-6) did not support virus replication, indicating the restriction of B19 virus replication to the erythroid cell lineage. Second, hematopoietic progenitor cells were serially assayed in B19-infected and uninfected bone marrow cultures. At initiation, B19 virus infection caused marked and moderate reduction in colony-forming unit erythroid (CFU-E) and burst-forming unit erythroid (BFU-E) numbers, respectively, without affecting CFU-Mix and CFU-GM numbers. Interestingly, the recovery of the erythroid progenitor numbers was observed at a late stage of cultures despite the sustained reduction in erythroblasts. The cells in the bursts derived from such reappearing BFU-E did not contain the virus genome. Although infectious virus was detected in the culture supernatants, the cultured CFU-E harvested at day 5 was relatively resistant to B19 virus infection compared with the CFU-E in fresh bone marrow. These findings suggest that pluripotent stem cells escaped B19 virus infection and restored the erythroid progenitor cells later in infected cultures. We conclude that the target cells of B19 virus are in the erythroid lineage from BFU-E to erythroblasts, with susceptibility to the virus increasing along with differentiation. Furthermore, the suppression of erythropoiesis and the subsequent recovery of the erythroid progenitor numbers in B19-infected liquid cultures may be analogous in part to the clinical features of B19 virus-induced transient aplastic crisis.     

In vitro interactions between thymocytes and hemopoietic precursor cells

Summary The effects of thymocytes on in vitro hemopoiesis were evaluated by co-culturing mixtures of mouse marrow cells and syngeneic thymocytes for granulocyte/macrophage precursors (CFU-GM), and for early (BFU-E) and late (CFU-E) erythroid precursors. Co-cultures were studied which contained fixed numbers of marrow cells with increasing numbers of thymocytes as well as co-cultures in which the total number of cells was kept constant. In addition, the effects of thymocytes on cultures stimulated by optimal or suboptimal amounts of humoral factors, the effects of a liquid phase incubation of the marrow cells with thymocytes prior to semi-solid culture, and the effects of irradiated and disrupted thymocytes were studied.The growth of CFU-GM was stimulated by thymocytes less than the growth of the other progenitor cells studied; at most, minimal stimulation by thymocytes was detected and at some marrow cell: thymocyte (M:T) ratios inhibition was even suggested. The growth of BFU-E was more strongly and more consistently stimulated than that of CFU-E. Killed thymocytes did not effect stimulation of CFU-E or BFU-E.These studies suggest that, in unfractionated marrow cells, the various hemopoietic precursor cells differ in their sensitivities to in vitro stimulation by thymocytes. In addition, it appears that only viable thymocytes can effect stimulation of the hemopoietic precursors in the vitro culture systems used.     

Role of endothelial cells in human hematopoiesis: modulation of mixed colony growth in vitro

The identification of clonal human multipotent hematopoietic progenitors has permitted an analysis of the growth factor requirements for these cells. Human endothelial cell cultures were used to examine the effects of media conditioned by the endothelial cells on human multipotent (CFU-mix) and committed erythroid (BFU-E, CFU-E) and myeloid (CFU-GM) precursors. These studies demonstrate that endothelial cells produce proteins of approximately 30,000 daltons, with isoelectric focusing points of 4.5 and 7.2, which stimulate the growth of human BFU-E and CFU-mix. A heat-labile protein(s) of 30,000 and 15,000 daltons stimulated the proliferation and differentiation of granulocyte-macrophage (CFU-GM) colonies. No erythropoietin was detected in endothelial cell supernatants. This suggests that endothelial cells, a normal component of marrow stroma, play an active role in the modulation of human hematopoietic stem cell growth.     

HLA-DR monoclonal antibodies inhibit the proliferation of normal and chronic granulocytic leukaemia myeloid progenitor cells

S ummary . We studied the capacity of murine monoclonal antibodies with HLA-DR specificity to inhibit the proliferation in vitro of erythroid (BFU-E and CFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells in normal bone marrow and the blood of patients with chronic granulocytic leukaemia (CGL). Two IgG₂ antibodies (CA 2.06 and L243) inhibited the proliferation of normal BFU-E and CFU-GM at relatively high dilution; a third antibody, DA2, had no effect on either progenitor cell. A complement-king monoclonal antibody with T-cell activity (OKT3) produced only minor reduction in progenitor cell proliferation. Further studies with L243 showed that BFU-E and CFU-GM from the blood of patients with CGL were inhibited to the same degree as normal marrow progenitor cells. The inhibition of progenitor
cell proliferation by a given antibody was always complement dependent and was therefore presumed to be due to a direct cytotoxic effect. The inhibitory effect of monoclonal antibodies is a valuable approach to the characterization of antigenic determinants on myeloid progenitor cells and the differential cytotoxicity of selected monoclonal antibodies might be exploitable for therapy.     

Modulation of erythropoiesis and myelopoiesis by exogenous erythropoietin in human long-term marrow cultures

In spite of their ability to support myelopoiesis for several months, human long-term marrow cultures (LTMC) are unable to sustain the production of mature erythroid cells for greater than 4 weeks. Because this preference correlates with the presence of myeloid growth factors and possible absence of erythroid factors in LTMC, we studied the effects of the erythroid growth and differentiation factor erythropoietin (Epo) on both erythropoiesis and myelopoiesis in human LTMC. Either natural or recombinant Epo was added weekly to LTMC for 10 weeks, and total cell number, numbers of hemopoietic progenitors (mixed lineage colony-forming units, CFU-MIX; erythroid burst-forming units, BFU-E; erythroid CFU, CFU-E; granulocyte-macrophage CFU (CFU-GM); granulocyte CFU, CFU-G; and macrophage CFU, CFU-M), erythroblasts (early and late), granulocytes, and macrophages were quantitated separately in the adherent and nonadherent layers of the cultures. In the absence of Epo, mature erythroid cells disappeared within the first 3-4 weeks, whereas in cultures supplemented with Epo, erythropoiesis was supported for up to 8 weeks. Results indicate that erythroid maturation is blocked at the BFU-E stage and that exogenous Epo may act on a mature subpopulation of BFU-E located in the nonadherent fraction of the cultures, promoting its maturation into CFU-E, which in turn develop into erythroblasts. However, despite Epo supplementation, erythropoiesis was not restored to in vivo proportions, suggesting that additional factors or conditions necessary for erythropoiesis are lacking in LTMC. Interestingly, we found that exogenous Epo reduced the numbers of presumably more mature (nonadherent) myeloid CFU (CFU-C), granulocytes, and macrophages compared to controls and did not alter the levels of any of the most primitive hemopoietic progenitors measured (CFU-MIX, adherent BFU-E, and adherent CFU-C). Thus the data show that exogenous Epo modulates hemopoiesis in human LTMC, enhancing erythropoiesis and suppressing myelopoiesis, but that its effects appear limited to modulating levels of the nonadherent (more mature) progenitors, leaving the numbers of the adherent (immature) progenitor cells unchanged.     

Effects of immunosuppressants, FK506, deoxyspergualin, and cyclosporine A on immature human hematopoiesis

Effects of the immunosuppressants, FK506, deoxyspergualin (DSG), and cyclosporine A (CsA) on the growth of human hematopoietic progenitor cells were tested in the presence of interleukin-3 (IL-3) with purified bone marrow and blood cells as targets in methylcellulose culture. FK506 had a significant stimulatory effect on the growth of colony- forming units/granulocyte-macrophage (CFU-GM) and burst-forming units/erythroid (BFU-E) from peripheral blood and cord blood cells but not from bone marrow cells. Neither DSG nor CsA had an effect on any type of target cell. Liquid-suspension-limiting dilution assay with IL- 3 showed that FK506 directly stimulated the growth of blood progenitors in a dose-dependent manner with single-hit kinetics. Liquid-suspension preincubation of blood cells with FK506 before culture in methylcellulose induced a significant increase in the amount of IL-3- supported growth of CFU-GM and BFU-E, whereas initial preincubation with IL-3 and subsequent culture with FK506 plus IL-3 exerted its stimulatory effect only on BFU-E. These data suggest that the stimulation of hematopoietic progenitor cells by FK506 occurs at a very early stage of maturation and diminishes with further myeloid development.     

Expression of CD11, CDw15, and transferrin receptor antigens on human hematopoietic progenitor cells

The expression of transferrin receptor-associated antigens and of CD11 and CDw15 antigens was investigated on myeloid committed progenitor cells (CFU-GM day 10, CFU-GM day 7, and cluster-forming cells [CFC] day 4), on erythroid committed progenitor cells (BFU-E and CFU-E), and on multilineage progenitor cells (CFU-GEMM). Both complement-dependent cytotoxicity and fluorescence-activated cell-sorting assays were performed. Complement-dependent cytotoxicity appeared to be the more sensitive assay. Transferrin receptor-associated antigens appeared to be clearly present on all myeloid and erythroid committed progenitor cells, but were found to be only weakly expressed on CFU-GEMM. CD11 antigens appeared to be strongly expressed only on mature granulocytes, monocytes, and certain lymphocytes, but not significantly on myeloid committed precursor cells. Surprisingly, CD11 antigens were weakly, but significantly, present on CFU-E. CDw15 antigens appeared to be restricted to myeloid differentiation and were increasingly expressed from CFU-GM day 10 to CFC day 4. Thus, antitransferrin receptor, CD11, and CDw15 antibodies can be used to separate hematopoietic progenitor cells and may be useful tools in the study of hematopoietic differentiation.     

Effect of recombinant human tumor necrosis factor on the colony growth of human leukemia progenitor cells and normal hematopoietic progenitor cells

The effects of recombinant human tumor necrosis factor (rH-TNF) on the colony growth of human leukemia progenitor cells (L-CFU), granulocyte- macrophage progenitor cells (CFU-GM), and erythroid progenitor cells (BFU-E) were studied. L-CFU was assayed with leukemia cells obtained from patients with acute myelogenous leukemia. CFU-GM and BFU-E were assayed with bone marrow cells obtained from hematologically normal donors and patients with acute leukemia or non-Hodgkin's lymphoma in complete remission. A dose-dependent growth inhibition of L-CFU as well as CFU-GM and BFU-E was observed by rH-TNF at concentrations of 1 to 100 U/mL. The inhibitory effect on L-CFU was significantly greater than that on CFU-GM. No correlation was observed between the inhibitory effect on L-CFU and the number of colonies formed in the cultures without rH-TNF. Preincubation of the progenitor cells in culture medium containing 20% fetal calf serum with up to 1,000 U/mL of rH-TNF for 24 hours did not result in the inhibition of colony growth of L-CFU or CFU- GM. The inhibitory effect of rH-TNF was neutralized by an anti-rH-TNF murine monoclonal antibody.     

Megakaryocytopoiesis in experimentally induced chronic normobaric hypoxia

It was recently proposed that prolonged hypoxia produces hypomegakaryocytic thrombocytopenia by reducing the pool of committed megakaryocyte progenitor cells at the expense of a greatly expanded erythroid progenitor pool. In order to test this hypothesis we have studied the relationship between megakaryocytopoiesis, erythropoiesis, and granulopoiesis at the level of progenitor cells (megakaryocyte colony-forming unit, CFU-Mk; erythroid CFU, CFU-E; erythroid burst-forming units; BFU-E; and granulocyte-macrophage CFU, CFU-GM) in the marrow of rats exposed for 4 weeks to normobaric hypoxia. We have found that hypomegakaryocytic thrombocytopenia was accompanied by decreased CFU-Mk, increased CFU-E, and a normal number of BFU-E and CFU-GM. These results support the hypothesis that prolonged hypoxia reduces the precursor cell commitment to differentiate into the megakaryocyte series by enhancing demand for differentiation into the erythroid cell line. However, the underlying mechanism needs further investigation.     

Effects of recombinant erythropoietin on the concentration and cycling status of human marrow hematopoietic progenitor cells in vivo

The concentration of human marrow progenitors CFU-E, BFU-E, CFU-GM, and CFU-Mk and the percentage of these progenitor cells in DNA synthesis were studied in nine patients with transfusion-dependent anemia of end- stage renal failure before and 2 weeks after treatment with human recombinant erythropoietin (Epo) at a dose of 150 to 300 U/kg intravenously three times per week. The concentration of CFU-E in the posttreatment marrow increased by a mean of 4.15-fold, BFU-E by 3.37- fold, CFU-GM by 1.86-fold, and CFU-Mk by 1.96-fold as compared with their respective concentrations in the pretreatment marrows. This increase in the concentrations of marrow progenitors was accompanied by almost a doubling of the percentage of these cells in DNA synthesis as assessed by the 3H-thymidine suicide technique. These observations demonstrate that at the progenitor cell level the human marrow responds to therapeutic doses of Epo as an organ rather than by a selective expansion of the erythroid cell line.     

Suppression of normal human erythropoiesis by human recombinant DNA-produced alpha-2-interferon in vitro

Interferons (IFN) have been shown to suppress the proliferation of human erythroid progenitors (BFU-E, CFU-E) in vitro. We have previously demonstrated that the inhibition of erythroid colony formation by gamma-IFN in vitro is mediated, in part, through the activation of monocytes and T-lymphocytes. In order to examine the mechanism(s) underlying the inhibitory action of one type of recombinant alpha-IFN (alpha-2-IFN) on erythropoiesis, the effect of different doses (80-10,000 U) of alpha-2-IFN on erythroid colony formation by normal human bone marrow cells in the presence or absence of monocytes and/or T cells was studied. The addition of alpha-2-IFN to whole marrow caused the suppression of BFU-E (10%-68%) and CFU-E (5%-75%) in a dose-dependent fashion. This inhibition occurred with the direct addition of alpha-2-IFN to culture plates but not with brief preincubation of marrow cells with alpha-2-IFN followed by washing of the cells. By contrast, brief exposure of marrow cells to gamma-IFN resulted in significant suppression of erythroid colony formation. The inhibitory action of alpha-2-IFN was not influenced by erythropoietin. Removal of monocytes and/or T cells prior to the addition of alpha-2-IFN failed to significantly reduce the suppressive effects of this molecule (BFU-E: 21%-66%; CFU-E: 20%-83%). Coculture of purified monocytes or T-lymphocytes preexposed to alpha-2-IFN with autologous bone marrow cells did not cause suppression of erythropoiesis; monocytes or T cells similarly treated with gamma-IFN, however, inhibited autologous BFU-E and CFU-E in vitro. These results demonstrate that, unlike gamma-IFN, the inhibitory effect of alpha-2-IFN on erythroid colony formation in vitro is not mediated to any significant degree through monocytes and T-lymphocytes. The suppressive effect of alpha-2-IFN occurs either directly at the erythroid progenitor(s) level and/or through accessory cell(s) other than monocytes and T cells.     

Agranulocytosis induced by propylthiouracil: evidence of a drug dependent antibody reacting with granulocytes, monocytes and haematopoietic progenitor cells

A patient with agranulocytosis and myeloid marrow hypoplasia following a second exposure to propylthiouracil (PTU) was studied for antibodies against mature blood cells and bone marrow precursor cells. During the acute phase of the agranulocytosis, significant growth inhibition of the myeloid committed progenitor cells (CFU-GM) was found following incubation with complement, indicating the presence of in-vivo cell bound cytotoxic antibodies. Using immunofluorescence and complement dependent cytotoxicity techniques it was demonstrated that acute phase and recovery phase sera contained circulating antibodies, reactive not only with differentiated granulocytes and monocytes but also with myeloid and erythroid (BFU-E/CFU-E) progenitor cells. Complement dependent lysis of the progenitor cells was facilitated by preincubation with PTU. These results indicate that the agranulocytosis was mediated by a PTU dependent antibody that affected both mature blood cells and bone precursor cells.     

Enrichment of progenitor cells from human marrow

Suspensions enriched for myeloid and erythroid colony-forming cells were prepared from human marrow by simultaneous treatment of low-density cells with the monoclonal antibodies Campath-1 and 80H.3, followed by immune-rosetting; lymphocytes and monocytes were successfully removed by this method. The final suspension contained 22% of blasts and 70% primitive myeloid-monocytic cells. GM-CFCs, counted after 14 days of culture, were enriched 21-fold. Of all cells present in the final suspension, 10.8% were day 8 GM cluster-forming cells, 2.3% were day 8 GM colony-forming cells, 2.4% were day 7 CFU-E, 0.9% were day 14 GM cluster-forming cells, 1.1% were day 14 GM colony-forming cells, and 0.6% were day 14 BFU-E. After enrichment, BFU-E became markedly more dependent on the addition of 5637-conditioned medium as a source of growth factors, suggesting that lymphocytes and/or monocytes support erythroid progenitor growth in cultures of unfractionated marrow. By removing these cells, we obtained a sensitive assay for burst-promoting activities in conditioned media. This procedure can be used to study the roles of marrow lymphocytes and monocytes in hemopoiesis as well as providing a basis for the purification of normal and aberrant progenitors.     

Severe in vitro inhibition of erythropoiesis and transient stimulation of granulopoiesis after bone-marrow infection with eight different HIV-2 isolates.

OBJECTIVE: To correlate severe anaemia and frequent neutropenia in HIV-2-infected patients with an inhibitory effect on bone-marrow progenitors common to several HIV-2 isolates. DESIGN: The effects of eight HIV-2 isolates on early (BFU-E) and late (CFU-E) erythroid progenitors and on granulomonocytic (CFU-GM) progenitors, produced in long-term bone-marrow cultures (LTBMC), were studied. METHODS: Absolute numbers of BFU-E, CFU-E and CFU-GM per culture flask were calculated weekly for each HIV-2-infected LTBMC using semi-solid clonogenic assays, and compared with those obtained in mock-infected LTBMC. Levels of significance for comparisons were determined by an analysis of variance (ANOVA). RESULTS: Pooled data from 24 series of LTBMC (three series for each HIV-2 isolate) revealed 80 and 100% inhibition of BFU-E and CFU-E on days 6 and 12 of LTBMC, respectively, while transient stimulation of CFU-GM was observed between days 14 and 20 of LTBMC, followed by total inhibition on day 30. CONCLUSIONS: These results confirm a direct inhibitory effect of HIV-2 on in vitro haematopoiesis. The similar pattern of erythroid progenitor inhibition obtained from seven out of eight isolates suggests that the inhibitory effect on erythropoiesis is a feature common to a large number of HIV-2 isolates, which correlates with clinical findings.     

Microenvironmental toxicity of azidothymidine: partial sparing with hemin

Azidothymidine (AZT) is a useful drug in management of AIDS. Nevertheless, its hematologic toxicity such as anemia and neutropenia present further complications to an already compromised hematopoietic state in patients. We studied the effects of AZT on human and murine bone marrow (BM) colony growth as determined by assays of CFU-E, BFU-E, CFU-GM, and fibroblastoid stromal (CFU-Fb) colonies. Cultures were grown in methylcellulose with growth factors and scored after three- to 14-day incubation. In general, murine marrow cultures were more sensitive to AZT as compared with human marrow. Furthermore, interindividual variation in toxicity to AZT was observed between marrow samples; 1 mumol/L AZT inhibited murine CFU-E, BFU-E, and CFU-GM by 98% to 100%, whereas human marrow was inhibited by 52%, 87%, and 65%, respectively. Lower concentrations of AZT (0.1 mumol/L) inhibited murine erythroid colony growth by 85% to 90%, whereas human growth was inhibited by only 39% to 52%. Myeloid colony inhibition was similar for human and murine systems. CFU-Fb growth was markedly suppressed (75%) by 1 mumol/L AZT. Hemin, at a concentration of 10 mumol/L, overcame some of the inhibitory effects of 1 to 0.1 mumol/L AZT without hindering antiviral activity. Inhibition of human CFU-E growth was completely overcome with hemin, whereas CFU-GM growth was recovered to 66% to 74% of control. A similar but less pronounced effect was observed for BFU-E. Furthermore, hemin does not decrease AZT's effects of HIV antigen content in vitro. We conclude that anemia and neutropenia, occurring as a result of AZT, may not be as pronounced in the presence of hemin. Furthermore, CFU-Fb was significantly reduced in the presence of low concentrations of AZT. This may indicate a major target site for BM toxicity since the stromal microenvironment may be responsible for maintaining short- and long-term hematopoiesis.     

Microenvironmentally dependent effects on murine haemopoiesis by a prolonged interleukin-1 treatment

Summary. We administered recombinant human IL-lβ (400 ng/d, s.c.) for 10 d to normal C57B1 mice and determined daily granuloid and erythroid parameters in marrow, spleen and blood.
In the marrow CFU-GM numbers were not affected but later granuloid cell stages were moderately enhanced (170%). In the spleen, however, CFU-GM numbers were sharply increased (1600%). whereas the granuloid precursors only doubled. Blood granulocytes increased transiently to 275% on day 5. In the marrow all erythroid parameters were severely reduced. This reduction was partially compensated by the spleen where initially only BFU-E and with some delay also more mature erythroid cells accumulated. At the end of the treatment mice were slightly anaemic. When mice were treated with IL-1 and erythropoietin (10 U/d) simultaneously, the inhibitory effects on erythropoiesis were less severe. In agreement with in vivo results, IL-1 inhibited in vitro colony growth of CFU-E from normal bone marrow and spleen but spleen CFU-E from 5 d IL-1 treated mice were insensitive.
We conclude that IL-1 can induce stimulation or inhibition of haemopoietic progenitor cells depending on their micro-environment.     

Bcl-2 Family Members As Prognostic Indicators in AML

It has been postulated that interleukin-lα (IL-lα) and interleukin-1β (IL-1β) play a role in the pathogenesis of the anemia of chronic disease by inhibiting the proliferation of human erythroid progenitor cells. In the course of investigating this hypothesis we found that IL-1 type I receptor (IL-1R) mRNA is expressed on erythroid progenitor enriched, primitive human hematopoietic cells (CD34+, c-kit-R^bright) and on cells isolated from human erythroid burst forming colonies (BFU-E). Nevertheless, when CD34+, c-kit-R^bright cells were exposed to IL-1α and IL-1β in vitro, cloning efficacy of BFU-E and CFU-E in a serum free culture system was not inhibited. Moreover, in apparent contradiction to the hypothesis being tested, we found that both IL-1 isoforms actually increased the survival of human BFU-E in serum free, growth factor free medium. Accordingly, these results suggest that if IL-1 plays a role in the pathogenesis of the anemia of chronic disease, it is not due to a direct suppressive effect on erythroid cell growth. Rather, our data support the hypothesis that IL-1 may cause the elaboration of another inhibitory cytokine (s) by cells of the marrow microenvironment.     

An analysis of the multilineage production of human hematopoietic progenitors in long-term bone marrow culture: evidence that reactive oxygen intermediates derived from mature phagocytic cells have a role in limiting progenitor cell self-renewal

To better understand the limited hematopoietic life span of human marrow "Dexter" cultures, we developed a miniaturized, two-stage culture system with which in vitro production of hematopoietic progenitors could be reproducibly detected and quantified. Light- density, gradient-separated human marrow cells were inoculated into Leighton slide tubes, and adherent ("stromal") cell layers were allowed to develop on the removable coverslips within these tubes during an initial 4 weeks of culture. Once stromal cell layers were established, cultures were irradiated (800 cGy) to eliminate all residual hematopoietic progenitors. The cultures were then recharged with autologous, cryopreserved marrow cells (enriched for BFU-E and CFU-GM) to reconstitute stem cell populations and to initiate in vitro hematopoiesis. Most progenitor cells added to irradiated cultures were no longer detectable by clonal assays within one to four days after recharge. Nonetheless, stable populations of adherent BFU-E and CFU-GM became established in these cultures within 24 to 48 hours, and when the total numbers of progenitors (adherent and nonadherent) were measured at weekly intervals thereafter, it was evident that both BFU-E and CFU-GM were generated in vitro. However, progenitor cell production declined as neutrophils and macrophages accumulated in the cultures. Moreover, with this accumulation of mature myeloid cells, increasing levels of O2- and H2O2 could be detected in the cultures, and it was found that the addition of oxidant scavengers (catalase and mannitol) to culture media enhanced the weekly expansions of progenitor cell numbers that could be measured. These findings support the conclusion that reactive O2 intermediates generated by mature myeloid cells have a role in limiting the duration and extent of hematopoietic progenitor cell self-renewal in long-term "Dexter" cultures of human marrow.