Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.

We previously described a method for isolating murine hematopoietic stem cells capable of reconstituting lethally irradiated recipients, which depends solely on dual-wavelength flow cytometric analysis of murine bone marrow cells stained with the fluorescent DNA-binding dye Hoechst 33342. This method, which appears to rely on the differential ability of stem cells to efflux the Hoechst dye, defines an extremely small and homogeneous population of cells (termed SP cells). We show here that dual-wavelength analysis of Hoechst dye-stained human, rhesus and miniature swine bone marrow cells reveals a small, distinct population of cells that efflux the dye in a manner identical to murine SP cells. Like the murine SP cells, both human and rhesus SP cells are primarily CD34-negative and lineage marker-negative. In vitro culture studies demonstrated that rhesus SP cells are highly enriched for long-term culture-initiating cells (LTC-ICs), an indicator of primitive hematopoietic cells, and have the capacity for differentiation into T cells. Although rhesus SP cells do not initially possess any hematopoietic colony-forming capability, they acquire the ability to form colonies after long-term culture on bone marrow stroma, coincident with their conversion to a CD34-positive phenotype. These studies suggest the existence of a hitherto unrecognized population of hematopoietic stem cells that lack the CD34 surface marker classically associated with primitive hematopoietic cells.

Essential role for the p55 tumor necrosis factor receptor in regulating hematopoiesis at a stem cell level.

Hematopoietic stem cell (HSC) self-renewal is a complicated process, and its regulatory mechanisms are poorly understood. Previous studies have identified tumor necrosis factor (TNF)-alpha as a pleiotropic cytokine, which, among other actions, prevents various hematopoietic progenitor cells from proliferating and differentiating in vitro. However, its role in regulating long-term repopulating HSCs in vivo has not been investigated. In this study, mice deficient for the p55 or the p75 subunit of the TNF receptor were analyzed in a variety of hematopoietic progenitor and stem cell assays. In older p55(-/-) mice (>6 mo), we identified significant differences in their hematopoietic system compared with age-matched p75(-/-) or wild-type counterparts. Increased marrow cellularity and increased numbers of myeloid and erythroid colony-forming progenitor cells (CFCs), paralleled by elevated peripheral blood cell counts, were found in p55-deficient mice. In contrast to the increased myeloid compartment, pre-B CFCs were deficient in older p55(-/-) mice. In addition, a fourfold decrease in the number of HSCs could be demonstrated in a competitive repopulating assay. Secondary transplantations of marrow cells from primary recipients of p55(-/-) marrow revealed impaired self-renewal ability of p55-deficient HSCs. These data show that, in vivo, signaling through the p55 subunit of the TNF receptor is essential for regulating hematopoiesis at the stem cell level.

Human recombinant granulocyte-macrophage colony-stimulating factor: a multilineage hematopoietin.

Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was tested for its ability to induce colony formation in human bone marrow that had been enriched for progenitor cells. In addition to its expected granulocyte-monocyte colony-stimulating activity, the recombinant GM-CSF had burst-promoting activity for erythroid burst-forming units and also stimulated colonies derived from multipotent (mixed) progenitors. In contrast, recombinant erythroid-potentiating activity did not stimulate erythroid progenitors. The experiments prove that human GM-CSF has multilineage colony-stimulating activity.

Interleukin 1 induces cultured human endothelial cell production of granulocyte-macrophage colony-stimulating factor.

Monokine-stimulated endothelial cells are known to produce both burst- and colony-stimulating activities, but neither the nature of the monokine nor the hematopoietic growth factor(s) produced is known. We show by mRNA analysis that an immortalized line of human endothelial cells constitutively produce granulocyte-macrophage colony-stimulating factor. Furthermore, interleukin 1 and tumor necrosis factor induce early passage human umbilical endothelial cells to produce the same growth factor.

Human recombinant granulocyte-macrophage colony-stimulating factor increases cell-to-cell adhesion and surface expression of adhesion-promoting surface glycoproteins on mature granulocytes.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to inhibit migration of mature granulocytes and to enhance their antibody-dependent cellular cytotoxicity. We found that human recombinant GM-CSF also enhanced granulocyte-granulocyte adhesion and increased by two- to threefold the surface expression of Mo1 and LeuM5 (P150, 95), two members of a family of leukocyte adhesion molecules (Leu-CAM). Increased Mo1 surface expression occurred within 15 min at 37 degrees C and was maximal at the migration inhibitory concentration of 500 pM. One-half maximal rise in the expression of Mo1 on the cell surface occurred at 5 pM. The chemotactic peptide f-Met-Leu-Phe produced a comparable rise in surface Mo1 with one-half maximal expression occurring at 7 nM. Both GM-CSF and f-Met-Leu-Phe produced optimal granulocyte-granulocyte adhesion at 500 pM and 100 nM, respectively. This adhesion-promoting effect induced by either stimulus was inhibited by a mouse monoclonal antibody directed against Mo1 antigen. These data indicate that GM-CSF promotes cell-to-cell adhesion, presumably through enhanced expression of leukocyte adhesion molecules. This mechanism may explain, in part, the known effects of GM-CSF on the function of mature granulocytes.

Dependence of highly enriched human bone marrow progenitors on hemopoietic growth factors and their response to recombinant erythropoietin.

Human bone marrow cells were sequentially fractionated by three negative selection steps to remove adherent cells and Fc receptor-bearing cells, followed by immune adsorption (panning) to deplete maturing cells that react with a panel of monoclonal antibodies. This nonadherent Fc receptor and antibody negative fraction could be further enriched by a positive selection "panning" step, using an antibody to HLA-DR antigen; 12-27% of the cells formed erythroid burst-forming unit (BFU-E), erythroid colony-forming unit, granulocyte-monocyte colony-forming unit, and erythroid and granulocyte and/or monocyte colony-forming unit-derived colonies with recovery of 0.5-1% of the cells and 20-100% of the colony-forming cells. Sequential fractionation resulted in increasing dependence of a subset of BFU-E-derived colonies on exogenous burst-promoting activity (BPA) for proliferation in culture, but the most enriched progenitor fraction still contained a proportion of accessory cell or BPA-independent BFU-E that responded to either natural or biosynthetic erythropoietin when added to cultures on day 0 in the absence of BPA. If the addition of erythropoietin was delayed until day 3, the data suggest that this population of BFU-E either died or became unresponsive to erythropoietin. Delayed addition of erythropoietin to cultures of enriched progenitors provided a sensitive BPA assay, since BPA-independent but erythropoietin-responsive BFU-E were eliminated. The surviving BFU-E that were dependent for their proliferation on the presence of both BPA and erythropoietin showed a characteristic dose response to increasing BPA concentrations.

Selective transgene expression for detection and elimination of contaminating carcinoma cells in hematopoietic stem cell sources.

Tumor contamination of bone marrow (BM) and peripheral blood (PB) may affect the outcome of patients receiving high dose chemotherapy with autologous transplantation of hematopoietic stem cell products. In this report, we demonstrate that replication defective adenoviral vectors containing the cytomegalovirus (CMV) or DF3/MUC1 carcinoma-selective promoter can be used to selectively transduce contaminating carcinoma cells. Adenoviral-mediated reporter gene expression in breast cancer cells was five orders of magnitude higher than that found in BM, PB, and CD34+ cells. Our results demonstrate that CD34+ cells have low to undetectable levels of integrins responsible for adenoviral internalization. We show that adenoviral-mediated transduction of a reporter gene can detect one breast cancer cell in 5 x 10(5) BM or PB cells with a vector containing the DF3/MUC1 promoter. We also show that transduction of the HSV-tk gene for selective killing by ganciclovir can be exploited for purging cancer cells from hematopoietic stem cell populations. The selective expression of TK followed by ganciclovir treatment resulted in the elimination of 6-logs of contaminating cancer cells. By contrast, there was little effect on CFU-GM and BFU-E formulation or on long term culture initiating cells. These results indicate that adenoviral vectors with a tumor-selective promoter provide a highly efficient and effective approach for the detection and purging of carcinoma cells in hematopoietic stem cell preparations.

Purification of fetal hematopoietic progenitors and demonstration of recombinant multipotential colony-stimulating activity.

To facilitate the direct study of progenitor cell biology, we have developed a simple and efficient procedure based upon negative selection by panning to purify large numbers of committed erythroid and myeloid progenitors from human fetal liver. The nonadherent, panned cells constitute a highly enriched population of progenitor cells, containing 30.4 +/- 13.1% erythrocyte burst forming units (BFU-E), 5.5 +/- 1.9% granulocyte-macrophage colony forming units (CFU-GM), and 1.4 +/- 0.7% granulocyte-erythroid-macrophage-megakaryocyte colony forming units (CFU-GEMM) as assayed in methylcellulose cultures. These cells are morphologically immature blasts with prominent Golgi. This preparative method recovers 60-100% of the committed progenitors detectable in unfractionated fetal liver and yields 2-30 X 10(6) progenitors from each fetal liver sample, and thus provides sufficient numbers of enriched progenitors to allow direct biochemical and immunologic manipulation. Using this technique, a purified recombinant protein previously thought to have only granulocyte-macrophage colony stimulating activity (GM-CSA) is shown to have both burst promoting activity and multipotential colony stimulating activity. Progenitor purification by panning thus appears to be a simple, efficient method that should facilitate the direct study of committed hematopoietic progenitors and their differentiation.

Stimulation of human hematopoietic colony formation by recombinant gibbon multi-colony-stimulating factor or interleukin 3.

Recently, the gene for a novel mammalian hematopoietic growth factor homologous to murine interleukin 3 was isolated from a gibbon T cell line and expressed in monkey COS cells. The factor, termed multi-colony stimulating factor (multi-CSF) or interleukin 3, is stimulatory to human target cells. We investigated the range of enriched human bone marrow and fetal liver hematopoietic progenitors responsive to multi-CSF; compared the colony types observed with those obtained in the presence of recombinant granulocyte-macrophage CSF (GM-CSF); and analyzed the effects on colony formation of combining multi-CSF with GM-CSF or granulocyte-CSF (G-CSF). The results show that multi-CSF acts as a multipoietin. Alone it stimulates the formation of colonies derived from granulocyte, macrophage, eosinophil, and megakaryocyte progenitors. In combination with erythropoietin it supports the development of both erythroid and mixed colonies. Furthermore, the data show that multi-CSF is a more potent stimulus of erythroid progenitors than GM-CSF. In combination with G-CSF multi-CSF substantially increases granulocyte colony number over the number obtained with each factor alone. We conclude that multi-CSF may prove to have important therapeutic potential in vivo as a stimulus for hematopoiesis.

Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

Cellular immune defects to Epstein-Barr virus-determined antigens in young males.

Three males with the X-linked lymphoproliferative syndrome (XLP) with hypo- or agammaglobulinemia following Epstein-Barr virus (EBV) infection and two males with the chronic mononucleosis syndrome were investigated for immune responses to EBV-determined antigens. Males with XLP showed profound cellular immune defects. Markedly diminished responses of natural killer cell and interferon-activated killer cell activities and impaired leukocyte migration inhibition responses to phytohemagglutinin were determined in patients with XLP. The two patients with chronic mononucleosis showed less severe defects. All patients showed partial or complete impairment of their EBV-specific immune responses as measured by leukocyte migration inhibition. EBV-specific antibodies were markedly diminished against EBV-associated nuclear antigen, early antigen, and viral capsid antigen in males with XLP. In contrast, patients with chronic mononucleosis had elevated antibodies to most EBV-specific antigens. Individuals with life-threatening EBV-induced lymphoproliferative disorders may exhibit multiple defective immune mechanisms against the virus.

Documentation of Epstein-Barr virus infection in immunodeficient patients with life-threatening lymphoproliferative diseases by clinical, virological, and immunopathological studies.

Multiple methods, pedigree analysis, clinical evaluation, and Epstein-Barr virus (EBV)-specific serology, EBV DNA hybridization of tissues to probe for viral genome, staining of touch imprints for EBV nuclear-associated antigen, establishment of spontaneous infected B-cell lines from peripheral blood or tissues, examination of peripheral blood smears, and hematopathology studies, were used to study seven patients with the X-linked lymphoproliferative syndrome and seven additional patients with life-threatening EBV-associated diseases. These studies demonstrated EBV in the tissues of all 14 patients and immunodeficient antibody responses to EBV were documented. This virus can produce various life-threatening lymphoproliferative diseases in a variety of immunodeficient patients.

Identification of distinct elements of the stromal microenvironment that control human hematopoietic stem/progenitor cell growth and differentiation.

Using a novel collection of conditionally immortalized mouse stromal cell clones, we evaluated the role of distinct elements of the hematopoietic microenvironment in supporting and regulating the growth, division, and differentiation of a candidate human stem cell population (CD34+/CD38-). We found functional diversity in the capacity of different stromal cell clones to support the growth of primitive (CD34+/CD38-) and committed (CD34+/CD38+) hematopoietic progenitors and their differentiation into mature hematopoietic cells (CD34-/CD45+). Among the stromal cell clones that supported long-term hematopoiesis, we identified two clones that induced expansion of CD34+ progenitor/stem cells during the first 4 weeks of coculture and that supported the maintenance of this CD34+ population for up to 10 weeks in vitro. However, these two clones appeared to represent two different microenvironments with regard to the signals they provide to the different CD34+ progenitor subpopulations: One stromal clone preserved a pool of undifferentiated, relatively quiescent (CD34+/CD38-) progenitor cells, allowing their differentiation at a low rate into more committed (CD34+/CD38+) progenitors; the other fostered a more extensive and rapid differentiation of all CD34+/CD38- progenitors into CD34+/CD38+ cells, preferentially maintaining this committed population at a higher rate of cell division. These stromal cell clones were also able to support the proliferation and differentiation of CD34+/CD38- cells in conditions in which progenitor-stroma contact was prevented. This collection of stromal cell clones may represent a unique tool for the study of stromal regulators of hematopoiesis as well as for the support of gene transfer into hematopoietic progenitor cells.

Decreased hematopoietic accessory cell function following bone marrow transplantation.

Hematologic engraftment following bone marrow transplantation requires not only pluripotent stem cells but also functioning accessory cells whose trophic factors support the proliferation and differentiation of stem cells and progenitors to mature blood cells. To better understand the regulation of hematopoiesis following transplantation, we studied hemopoietic accessory cell function in bone marrow transplant recipients 3 weeks to 10 months following transplantation. In general, hematopoietic accessory cell function was decreased following bone marrow transplantation. Sequential fractionation of accessory cells demonstrated that adherent cells often produced near-normal functional burst-promoting activity (BPA) and granulocyte-macrophage colony-stimulating activity (GM-CSA), but Fc receptor-positive (Fc+) cells and T cells uniformly produced greatly reduced BPA and GM-CSA, as compared to transplant donor cells. This cellular deficiency was corrected by soluble burst-promoting activity and granulocyte-macrophage colony-stimulating activity and so appeared to be due to the failure of accessory cells to produce trophic hormones. Limiting-dilution analysis (LDA) for proliferating T-cell precursors demonstrated a greatly reduced frequency in phytohemagglutinin-responsive cells, supporting the role of deficient hematopoietic growth factor production by activated T cells in transplant recipients. This hemopoietic accessory cell defect may thus reflect more generalized lymphocyte dysfunction in these patients. Hematopoiesis following bone marrow transplantation appears to rely upon growth factors released by accessory cells in the adherent layer.

Synergistic effects of interleukin-11 with other growth factors on the expansion of murine hematopoietic progenitors and maintenance of stem cells in liquid culture.

The effect of interleukin-11 (IL-11), alone and in combination with other hematopoietic growth factors, was studied in liquid suspension cultures of murine bone marrow. Stem and progenitor cell contents of input bone marrow 2 days after 5-fluorouracil (5-FU) treatment (FU2dBM) and lineage-depleted, Sca-1-positive FU2dBM (Lin-Sca+Fu2dBM) were compared with output values obtained after 6 days in different growth factor combinations. Stem and progenitor cell recoveries were measured by long-term in vivo competitive repopulation, spleen colony-forming unit (CFU-S), and in vitro limiting dilution long-term bone marrow culture (cobblestone area-forming cell [CAFC]) assays. IL-11, IL-3, IL-6, and Steel factor (SF) were tested separately and in combinations of two, three, and four factors. IL-11 was incapable of maintaining the survival of stem or progenitor cells as a single agent but synergized with either IL-3 or SF to expand progenitor cell output in cultures of FU2dBM. IL-11 greatly enhanced progenitor cell expansion from FU2dBM when added to two- and three-factor combinations of SF, IL-3, and IL-6. The recovery of long-term repopulating ability was enhanced four-fold by incubation in SF plus IL-11 compared to SF alone. IL-11 in combination with SF, IL-3, and IL-6 stimulated a 24,000-fold expansion of progenitor cells from stem cell-enriched Lin-Sca+FU2dBM. Overall, the results indicate that IL-11 is a potent synergistic factor for stem cell proliferation and expansion of progenitors in liquid culture.

Membrane antigen expression during hemopoietic differentiation.

The pattern of certain groups of antigens expressed on the surface of hemopoietic cells changes either during the course of differentiation from pluripotent stem cells to mature functional cells or as a function of the proliferative state of the cells. A map of these changes is emerging and is providing valuable information for selecting and purifying rare stem cells and for classifying the acute leukemias. This knowledge is also beginning to provide insights into physiological and pathological cellular interactions affecting the early stages of hemopoiesis, and is being exploited to remove T lymphocytes from allogeneic bone marrow grafts in order to prevent graft-vs.-host disease as well as leukemic cells from bone marrow before autologous reinfusion. In this article I will briefly review the cellular basis of hemopoiesis and then discuss the methods used to determine the presence of antigens on normal hemopoietic cells. I will then summarize the pattern of membrane antigens expressed during differentiation and conclude by discussing the biological and therapeutic implications.

Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare, congenital, hypoplastic anemia that usually presents in early infancy. Congenital anomalies, particularly of the head and upper limbs, are present in about a quarter of reported patients. The disease is characterized by a moderate-to-severe macrocytic anemia, occasional neutropenia or thrombocytosis, a normocellular bone marrow with erythroid hypoplasia, and an increased risk of developing leukemia. The pathogenesis is unknown. The majority of patients respond to prednisone, and often erythropoiesis can be maintained with low doses of the drug. Both remissions and increased resistance to steroid treatment can occur. Nonresponders usually are transfusion dependent, although responses to high dose steroid, androgen, and interleukin-3 have been observed. Bone marrow transplantation can be curative.

[Regulation of hematopoiesis]. / Reguliatsiia krovetvoreniia.

Hematopoiesis is regulated by a tiny population of cells, the stem cells and committed progenitors. These cells are under the influence of hematopoietic growth factors most of which are produced immediately in their environments. Their fates are strongly influenced by those growth factors. The growth factors interact with specific receptors that are present on progenitor surfaces and may in fact induce changes in the levels of those receptors. The receptors form dimers or heterodimers to pass proliferative and differentiation signals into the cell. We do not yet know exactly how that signalling is accomplished, but will probably known much more about it in the next year or so.