Activity of the ligand for c-mpl, thrombopoietin, in early haemopoiesis

We examined the role of the ligand for c-mpl , thrombopoietin (TPO), in murine early haemopoiesis, using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) supported colony formation by marrow cells of 5-fluorouracil (5-FU)-treated mice, whereas TPO alone yielded no colony. When blast cell colonies grown in the presence of TPO plus SF or TPO plus IL-3 were individually replated in suspension cultures containing serum and several growth factors, various combinations of myeloid lineages were seen, indicating that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of haemopoietic progenitors. We then studied the effects of TPO on proliferative kinetics of cycling progenitors. TPO hastened IL-3-dependent growth of progenitors by shortening the time required for cell cycling. These results suggest that TPO, as a single factor, can support the survival of haemopoietic progenitors and TPO synergizes with SF or IL-3 to act on early multipotential haemopoietic progenitors.     

Activity of the ligand for c-mpl,thrombopoietin, in early haemopoiesis

We examined the role of the ligand for c-mpl, thrombopoietin (TPO), in murine early haemopoiesis, using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) supported colony formation by marrow cells of 5-fluorouracil (5-FU)-treated mice, whereas TPO alone yielded no colony. When blast cell colonies grown in the presence of TPO plus SF or TPO plus IL-3 were individually replated in suspension cultures containing serum and several growth factors, various combinations of myeloid lineages were seen, indicating that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of haemopoietic progenitors. We then studied the effects of TPO on proliferative kinetics of cycling progenitors. TPO hastened IL-3-dependent growth of progenitors by shortening the time required for cell cycling. These results suggest that TPO, as a single factor, can support the survival of haemopoietic progenitors and TPO synergizes with SF or IL-3 to act on early multipotential haemopoietic progenitors.     

Selective expression of class-II MHC antigens during hemopoietic differentiation

Human multipotential or "mixed" bone marrow colony-forming cells and lineage-restricted colony-forming progenitors have been analyzed by cell sorting (FACS) using a series of monoclonal antibodies. The latter all react with monomorphic class-II MHC glycoprotein determinants but differ in their reactivity or cross-reactivity with products of different loci, i.e., SB (DP), DC (DQ), DR. The results confirm that very few progenitor cells detectably express DC. Anti-DR monoclonals varied in their reactivity with progenitor cells. The majority of progenitors in all lineage categories express determinants that are shared or cross-reactive between DR and SB while fewer progenitors can be shown to bind antibodies specific for DR or SB.     

Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice

Recently, the ligand for the Mpl receptor (ML) was identified to be thrombopoietin, the principal regulator of megakaryocytopoiesis and thrombopoiesis. We examined the effects of ML, as a single factor or in combinations with early acting factors such as steel factor (SF), interleukin (IL)-3, IL-1, IL-6, and granulocyte colony-stimulating factor (G-CSF), on colony formation from primitive progenitors of mice. Cells enriched for cell cycle dormant primitive progenitors were isolated from bone marrow cells of 5-fluorouracil (5-FU)-treated mice by a combination of Nycodenz density gradient separation, immunomagnetic selection for lineage-negative cells, and fluorescence- activated cell sorter (FACS) sorting for Ly-6A/E+Kit+ cells. ML, in the presence of erythropoietin, could support the formation of only a few megakaryocyte colonies. However, ML acted synergistically with SF or IL- 3 to support the formation of multiple types of hematopoietic colonies including multilineage colonies. Effects of the combination of ML and SF on multipotential progenitors were not mediated through other cells, as demonstrated by micromanipulation of individual progenitors. In suspension culture, the combination of ML and SF increased the number of multipotential progenitors. ML also acted synergistically with IL- 11, IL-6, or G-CSF to support colony formation in serum-containing, but not in serum-free, cultures. However, the multilineage colony formation seen in serum-containing culture was completely abrogated by addition of ACK2, a neutralizing antibody to Kit protein. Serial observation (mapping studies) of colony development from multipotential progenitors suggested that ML triggers the cell division of dormant progenitors. Based on these observations, we propose that ML can function as an early acting cytokine and stimulate the proliferation of cell cycle dormant progenitors by shortening their G0 period.     

A possible change in doubling time of haemopoietic progenitor cells with stem cell development

We separated haemopoietic progenitors derived from marrow cells of 5-fluorouracil (5-FU)-treated mice into three groups, based on the stages of stem cell development and studied doubling time, using a serum-free clonal culture system. Stage I progenitors were those present in primary marrow cells from 5-FU-treated mice. Stages II and III progenitors were early and late progenies in culture of stage I progenitors, respectively. The morphological analysis of colonies derived from stage I, II and III progenitors demonstrated an association of progression of stages with loss of multipotentiality. The doubling time of haemopoietic progenitors was estimated by sequential analysis of colony formation and studies of growth fraction. The time required for haemopoietic progenitors to double shortened as their stage of development progressed. Alteration in one doubling time of haemopoietic progenitors at progressive stages of stem cell development was seen in cultures supported by various combinations of growth factors, including interleukin-3 (IL-3), IL-11, and steel factor (SF). Cell-cycle analysis suggested that reduction of the doubling time of haemopoietic progenitors is probably due to a decrease in the time spent in the G1 phase of the cell cycle. Our results suggest that in early haemopoiesis the doubling time of haemopoietic progenitors may change with stem cell development.     

Platelet-derived growth factor stimulates growth of highly enriched multipotent haemopoietic progenitors

Platelet-derived growth factor (PDGF) has been shown to stimulate growth of normal and malignant fibroblasts, glial cells and smooth muscle cells. A growth promoting effect on human haemopoietic precursors has also been described, but the interpretation of this haemopoietic proliferative response to PDGF has been hampered by the lack of purity of the target population. In this study we show that PDGF promotes growth of early bone marrow haemopoietic progenitors depleted of either monocytes or T lymphocytes which are known to influence haemopoiesis. Moreover, the action of PDGF is even increased on a highly enriched BI-3C5 early bone marrow population. BI-3C5 is a novel monoclonal antibody which recognizes an antigen present on all multilineage colony-forming cells (CFU-mix) (Tindle et al. 1985). BI-3C5 positively and negatively sorted fractions were obtained by fluorescence activated cell sorting (FACS) and PDGF was found to stimulate growth of CFU-mix in the BI-3C5-positive fraction (consisting of only 4-6% of the marrow population), the effect being more marked than that on unsorted bone marrow. The results suggest that the product of the cellular proto-oncogene c-sis (the putative structural gene for the beta chain of PDGF) may play a regulatory role in the in vivo proliferation of multipotent haemopoietic progenitors.     

A possible change in doubling time of haemopoietic progenitor cells with stem cell development

We separated haemopoietic progenitors derived from marrow cells of 5-fluorouracil (5-FU)-treated mice into three groups, based on the stages of stem cell development and studied doubling time, using a serum-free clonal culture system. Stage I progenitors were those present in primary marrow cells from 5-FU-treated mice. Stages II and III progenitors were early and late progenies in culture of stage I progenitors, respectively. The morphological analysis of colonies derived from stage I, II and III progenitors demonstrated an association of progression of stages with loss of multipotentiality. The doubling time of haemopoietic progenitors was estimated by sequential analysis of colony formation and studies of growth fraction. The time required for haemopoietic progenitors to double shortened as their stage of development progressed. Alteration in one doubling time of haemopoietic progenitors at progressive stages of stem cell development was seen in cultures supported by various combinations of growth factors, including interleukin-3 (IL-3), IL-11, and steel factor (SF). Cell-cycle analysis suggested that reduction of the doubling time of haemopoietic progenitors is probably due to a decrease in the time spent in the G1 phase of the cell cycle. Our results suggest that in early haemopoiesis the doubling time of haemopoietic progenitors may change with stem cell development.     

Effect of recombinant and purified human haematopoietic growth factors on in vitro colony formation by enriched populations of human megakaryocyte progenitor cells

Nonadherent low density T-lymphocyte depleted (NALT-) marrow cells from normal donors were sorted on a Coulter Epics 753 Dye Laser System using Texas Red labelled My10 and phycoerythrin conjugated anti HLA-DR monoclonal antibodies in order to obtain enriched populations of colony forming unit-megakaryocyte (CFU-MK). The CFU-MK cloning efficiency (CE) was 1.1 +/- 0.5% for cells expressing both high densities of My10 and low densities of HLA-DR (My10 DR+). This procedure resulted in an 18-fold increase in CE over NALT- cells. The effect of purified or recombinant human haematopoietic growth factors including erythropoietin (Epo), thrombocytopoiesis stimulating factor (TSF), interleukin 1 alpha (IL-1 alpha), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF or CSF-1) and interleukin MK colony formation by My10 DR+ cells was determined utilizing a serum depleted assay system. Neither Epo, TSF, CSF-1, IL-1 alpha nor G-CSF alone augmented MK colony formation above baseline (2.5 +/- 0.8/5 x 10(3) My10 DR+ cells plated). In contrast, the addition of GM-CSF and IL-3 each increased both CFU-MK colony formation and the size of colonies with maximal stimulation occurring following the addition of 200 units/ml of IL-3 and 25 units/ml of GM-CSF. At maximal concentration, IL-3 had a greater ability to promote megakaryocyte colony formation than GM-CSF. The stimulatory effects of GM-CSF and IL-3 were also additive in that the effects of a combination of the two factors approximated the sum of colony formation in the presence of each factor alone. The CFU-MK appears, therefore, to express HPCA-1 and HLA-DR antigens. These studies also indicate that GM-CSF and IL-3 are important in vitro regulators of megakaryocytopoiesis, and that these growth factors are not dependent on the presence of large numbers of macrophages or T cells for their activity since the My10 DR+ cells are largely devoid of these accessory cells.     

Irradiation enhances the support of haemopoietic cell transmigration, proliferation and differentiation by endothelial cells

Endothelial cells (ECs) are a critical component of the bone marrow stroma in the regulation of haemopoiesis. Recovery of bone marrow aplasia after radiation exposure depends, in part, on the repair of radiation-induced endothelial damage. Therefore, we assessed the ability of an irradiated human bone marrow EC line (TrHBMEC) to support transmigration, proliferation and differentiation of CD34+ bone marrow cells either irradiated or not in transendothelial migration or co-culture models. Radiation-induced EC damage was reflected by an increased release of soluble intercellular adhesion molecule (sICAM)-1 and platelet endothelial cell adhesion molecule (PECAM)-1. Irradiation of TrHBMECs with a 10 Gy dose strongly enhanced the transmigration of CD34+ cells, granulo-monocytic progenitors (CFU-GM) and erythroid progenitors (BFU-E). While ICAM-1 and PECAM-1 expression on irradiated TrHBMECs was increased, only antibodies against PECAM-1 inhibited the radiation-induced enhanced transmigration of haemopoietic cells. Irradiation of TrHBMECs (5-15 Gy) also increased proliferation and differentiation towards the granulo-monocytic lineage of co-cultured CD34+ cells, as well as colony formation by those cells and the production of interleukin 6 (IL-6), IL-8, granulocyte colony-stimulating factor (CSF) and granulocyte-macrophage CSF. Irradiated TrHBMECs were more capable of stimulating irradiated (1,2 Gy) CD34+ cells and haemopoietic progenitors than non-irradiated TrHBMECs. Together, these results suggest that, despite the radiation-induced damage, irradiated ECs may favour haemopoietic reconstitution after radiation exposure.     

The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence- activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38- , HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.     

Effect of interleukin 6 (IL-6) on the differentiation and proliferation of murine and human hemopoietic progenitors

We examined the effect of human recombinant (r) interleukin 6 (IL-6) on the differentiation of murine and human hemopoietic progenitors. Human IL-6 supported colony formation by murine bone marrow cells. These colonies consisted of neutrophils and macrophages. Recombinant IL-6 was able to support multilineage colony formation by spleen cells from 5-fluorouracil (5-FU)-treated mice. These colonies consisted of greater than 1 x 10(4) cells. Differential counts revealed large colonies exhibiting different combinations of cell lineages: neutrophils, macrophages, eosinophils, mast cells, and megakaryocytes. However, when blast cell colonies supported by interleukin 3 were replated into secondary dishes containing IL-6, they could differentiate into only neutrophils and macrophages. Single cells transferred from blast cell colonies formed only neutrophil/macrophage colonies. These results indicate that IL-6 had a direct effect on the growth and development of murine granulocyte-macrophage progenitors at a late stage and a significant effect on multipotential hemopoietic precursors that might be indirect through other cells. By contrast, human rIL-6 did not support colony formation by human bone marrow mononuclear cells. IL-6 may not show an independent activity for human hemopoiesis of myeloid lineage. However, the synergistic activity of IL-6 remains to be clarified.     

Selective enhancement of multipotential hematopoietic progenitors in vitro and in vivo by IL-20

In a search for novel growth factors, we discovered that human interleukin-20 (IL-20) enhanced colony formation by CD34+ multipotential progenitors. IL-20 had no effect on erythroid, granulocyte-macrophage, or megakaryocyte progenitors. IL-20 transgenic mice increased the numbers and cell cycling of multipotential but not other progenitors. IL-20 administration to normal mice significantly increased only multipotential progenitor cells, demonstrating that IL-20 significantly influences hematopoiesis, with specificity toward multipotential progenitors. This is the first cytokine with such specificity identified.     

Haemopoietic colony-forming cells from peripheral blood stem cells harvests: cytokine requirements and lineage potential

Summary. We have measured the in vitro growth requirements of progenitor cells released into the blood of cancer patients following administration of chemotherapy and cytokines. In order to distinguish the direct effects of cytokines on progenitors from those activating acessory cells, we have comparied clonogenic grwoth before and after CD34-positive selection of progenitors, in serum-free conditions. CD34 selection had little effect on the cytokine requirements of erythroid colony-forming cells and single cytokines, particularly interleukin-3, could support considerable colony growth in both mononulear and CD34+ cell suspensions. Optimal erythroid colony grwoth, however, usually required the addition of a combination of stem cells factor and interleukin-3, in addition to erythoropoietin, which was was always required. Maximal numbers of granulocyte monocyte progenitors in mononuclear cell cultures, could be achieved with a mixture of stem cell factros, ionterleukin-3 and granulocyte-monocyte colony stimulating factor. How ever, after CD34 selection, full myeloid colony growth was only achieved when granulocyte colony stimulating factor was added to the above mixture. This presumably reflects loss of accessory cells, during CD34 selection, which produced this cytokine. When transplanted after 8 d of culutre. 16/22 myeloid colonies from erythorpoietin-free cultures of peripheral blood stem cell harvests, could generate secondary multipotential. However, surface marker analysis of individual erythroid colonies revealed only the occasional presence of granulocytes and monocytes. These date demonstrate that cytokine mixtures are required for optimal colony growth, particuarly after CD34 selection, a nd that most mobilizied, blood clonogenic cells are multipotential.     

VLA-4 and VCAM-1 are the principal adhesion molecules involved in the interaction between blast colony-forming cells and bone marrow stromal cells

Summary. The molecular basis and functional significance of interactions between haemopoietic progenitor cells and the stromal microenvironment is still poorly understood. Here we investigated a broad panel of surface adhesion molecules for their involvement. For this purpose, the colony-forming capacity of stroma-adherent Bl-CFC, BFU-E and GM-CFC was studied. Both mononuclear bone marrow cells (BMC) and bone marrow-derived stromal cells (BMSC) express a wide variety of adhesion molecules. However, only antibodies against β1-, α4-integrin (both chains of the very late activation antigen-4 (VLA-4)) and vascular cell adhesion molecule (VCAM-1) inhibited colony formation from stroma-adherent Bl-CFC by 50% or more. Antibodies against a panel of other adhesion molecules, including the α5-integrin chain, were without effect. Subsequent pretreatment experiments revealed that VLA-4 on progenitors interacted with stromal VCAM-1. The inhibitory antibodies did not interfere with the clonogenic capacity of but with adhesion of BFU-E and GM-CFC. Whether the inhibitory antibodies act similarly on progenitors which depend on BMSC for growth and/or differentiation, such as Bl-CFC, remains to be determined.     

Synergism of interferon-gamma and stem cell factor on the development of murine hematopoietic progenitors in serum-free culture

We examined the effects of interferon-gamma (IFN-gamma) on the growth of murine hematopoietic progenitors supported by interleukin-3 (IL-3) or stem cell factor (SCF) in a serum-free culture system. IFN-gamma inhibited IL-3-dependent granulocyte-macrophage colony growth by normal bone marrow cells, but increased the number of pure and mixed megakaryocyte colonies by post-5-fluorouracil bone marrow cells. The addition of IFN-gamma to the culture containing SCF resulted in a synergistic action on the development of primitive hematopoietic progenitors as well as on the development of mature populations. Primitive progenitors responding to SCF + IFN-gamma were suggested to be supported by SCF in the early stage of development and require IFN- gamma for subsequent growth. Replating experiments of blast cell colonies and comparison of total colony growth among SCF + IFN-gamma, SCF + IL-3, and SCF + IFN-gamma + IL-3 suggest that multipotential progenitors supported by SCF + IFN-gamma are a part of those reactive to SCF + IL-3. These findings suggest that IFN-gamma has bifunctional activity on murine hematopoiesis.     

GROWTH FACTOR INDUCTION OF CYTOSOLIC PROTEIN TYROSINE KINASE ACTIVITY IN HUMAN HAEMOPOIETIC PROGENITOR CELLS ISOLATED BY FLOW CYTOMETRY

We employed a highly sensitive method to assay protein tyrosine kinase activity in extracts of subpopulations of CD34⁺ bone marrow progenitor cells isolated by fluorescence activated cell sorting in an attempt to better define how growth-factor induction of enzymatic activity relates to progenitor cell maturation. FACS analysis confirmed that, under the conditions employed, essentially all of the CD34⁺ cells in adult human marrow that lacked the CD38 antigen were devoid of the myeloid maturation marker CD33 as well as the lineage antigens: CD10, 13, 14, 15, 16, 19, 71 and glycophorin A. A variable portion (50–90%) of these CD34⁺, CD38^? progenitor cells expressed HLA-DR. CD34⁺, CD38^? cells that did not express HLA-DR were found to lack detectable levels of either membrane or cytosolic tyrosine kinase activity. HLA-DR⁺ progenitor cells that lacked CD38 possessed elevated levels of cytosolic tyrosine kinase activity but only low levels of plasma membrane activity. In contrast, CD34⁺ cells that expressed CD38 (and HLA-DR) possessed high levels of membrane-associated tyrosine kinase activity. A cocktail of haemopoietic growth factors that included IL-3, IL-6 and stem cell factor effectively induced tyrosine kinase activity in CD34⁺, CD38^?, HLA-DR^? progenitor cells. Growth factor induction of tyrosine kinase activity in these cells was not inhibited by actinomycin D or cyclohexamide. Most of the tyrosine kinase activity induced by these growth factors was recovered from the cytosolic fraction of disrupted cells. Thus, induction of cytosolic tyrosine kinase activity is an early event in the response of uncommitted haemopoietic cells to haemopoietic growth factors. Subsequent activation of membrane tyrosine kinases may initiate key transduction processes as these cells begin to differentiate.     

Purified interleukin-3 and erythropoietin support the terminal differentiation of hemopoietic progenitors in serum-free culture

We studied the effect of purified interleukin-3 (IL-3) and erythropoietin on colony formation by hemopoietic progenitors in serum- free cultures of spleen cells from 5-fluorouracil (5-FU)-treated mice. In the presence of IL-3 alone, most of the multilineage (three or more lineages) colonies did not contain erythroid cells. However, in the presence of IL-3 and erythropoietin, most of the multilineage colonies contained various numbers of erythroid cells. Replating experiments suggest that IL-3 maintains the growth of the progenitor cells, which could differentiate into erythroid cells. Erythropoietin facilitated the terminal differentiation and amplification of erythroid cells, although it did not sustain the growth of multipotential stem cells. Single-cell transfer experiments demonstrate that IL-3 supported the late stages of differentiation of neutrophils, macrophages, eosinophils, and megakaryocytes in the absence of lineage-specific factors. Therefore, IL-3 supports the differentiation of multilineage hemopoietic progenitors, and the terminal differentiation of most hemopoietic lineages, with the exception of the erythroid lineage.     

The myelosuppressive effect of recombinant interferon γ in short-term and long-term marrow cultures

The effect of a highly purified human gamma IFN (r-gamma-IFN) on the growth of haemopoietic progenitors was examined in soft agar assays and in long-term bone marrow cultures. r-gamma-IFN reduced colony formation by progenitor cells of the granulocyte/macrophage lineage (GM-CFC), the erythroid lineage (BFU-E) and multipotent cells (GEMM-CFC), and suppressed haemopoiesis in long-term culture in a dose-related fashion. At high doses (1000 units/ml) r-gamma-IFN appeared to be toxic to the stromal cells of the bone marrow.     

Changes in cell surface antigen expression during hemopoietic differentiation

Human bone marrow cells were separated on a fluorescence activated cell sorter (FACS) according to their binding of a series of monoclonal antibodies; the positive and negative fractions were cloned for erythroid burst and colony-forming units (BFU-E and CFU-E) and myeloid colony-forming units (CFU-GM), and cytocentrifuge slides were prepared for microscopy of maturing precursors. The pattern of antigen expression on hemopoietic progenitor and precursor populations has been established using antibodies defining blood group (A, I/i), HLA- associated (*A, B, C, DR, DC1), lineage specific, and transferrin receptor antigens. Like monomorphic HLA-DR, the antigen defined by monoclonal antibody OKT10 is expressed on the earliest progenitors and lost during differentiation, suggesting a role in interactions regulating the differentiation of these cells. The HLA-linked DC1 determinant, in contrast to HLA-DR, is not expressed at a detectable level on progenitor cells. Although a lineage-specific early antigen has not been identified, the transferrin receptor is expressed on the majority of erythroid progenitors, but only weakly on myeloid progenitors, and may provide an approach to isolating erythroid progenitors. These and earlier studies with monoclonal antibodies against HLA-DR and glycophorin now provide a detailed "map" of antigen expression during hemopoietic differentiation.     

Growth factor requirements for survival in G0 and entry into the cell cycle of primitive human hemopoietic progenitors.

In this study we have isolated populations of dormant human hemopoietic progenitors by two different approaches. First CD34+ cells isolated by panning were further separated on the basis of absence of HLA-DR expression by using fluorescence-activated cell sorting. Second, CD34+ HLA-DR- cells were isolated by nonadherence to soybean agglutinin, negative immunomagnetic bead selection with lineage-specific antibodies, and two-color cell sorting. Progenitors in either cell population were unable to form colonies in the presence of interleukin (IL)-3 alone but yielded a substantial number of colonies, including multilineage colonies, in the presence of combinations of IL-3 and IL-6. Similarly, IL-3 plus any one of the other synergistic factors, including granulocyte colony-stimulating factor, IL-11, leukemia inhibitory factor, and steel factor, effectively supported colony formation from CD34+ HLA-DR- progenitors. Sequential observation of colony formation from single CD34+ HLA-DR- cells provided definitive evidence that the synergistic factors trigger cell divisions of dormant cells. Studies with delayed addition of factors to the cultures provided evidence that this population of cells also requires IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) to survive even while dormant. In contrast, none of the synergistic factors were able to replace IL-3 or GM-CSF in this function. These findings confirm and extend the model that multiple factors with overlapping functions operate both independently and in combination to regulate early stages of hemopoiesis.