Survival Of Hematopoietic Progenitor Cells Research Articles

Effects of Interleukin-3 and c-kit Ligand on the Survival of Various Classes of Human Hematopoietic Progenitor Cells

AbstractWe examined the effects of interleukin-3 (IL-3) and c-kit ligand (KL) on the survival of differentiated hematopoietic progenitor cells (HPC), the burst-forming unit-erythroid (BFU-E); colony-forming unit-granutocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM); and CFU-granulo-cyte-monocyte (CFU-GM) and more primitive hematopoietic cells that give rise to these progenitor cells (pre-colony-forming cells [pre-CFC]). CD34+ HLA-DR+ cells, which are highly enriched for committed HPC, and CD34+ HLA-DR− c-kit+ cells, which contain the most primitive assayable hematopoietic cells, including long-term bone marrow culture-initiating cells, high proliferative potential-CFC, the CFU-blast, and the BFU-megakaryocyte, were suspended in serum-free medium in the presence or absence of IL-3 or KL. CD34+ HLA-DR+ cells incubated under serum-free conditions or in the presence of KL for 96 hours lost greater than 90% of assayable unilineage or multilineage HPC, whereas those cells incubated in the presence of IL-3 retained 40% of the number of HPC present at time 0. The effect of IL-3 on HPC survival was most pronounced on the BFU-E and CFU-GEMM present within CD34+ HLA-DR+ cells. Addition of IL-3, but not of KL, to CD34+ HLA-DR+ cells delayed the appearance of morphologic changes and DNA fragmentation patterns associated with cell death occurring by apoptosis. CD34+ HLA-DR− c-kit+ cells were incubated under similar serum-free conditions in the presence or absence of IL-3 or KL, and the frequency of pre-CFC was determined by limiting dilution analysis. The frequency of pre-CFC in cells incubated for 48 hours in the absence of serum was similar to that of cells incubated in the presence of IL-3 and approximately doubled when CD34+ HLA-DR− c-kit+ cells were incubated in the presence of KL. Addition of KL to serum-free suspension cultures of CD34+ HLA-DR− c-kit+ cells delayed the appearance of DNA fragmentation patterns associated with apoptosis to a greater extent than did the addition of IL-3. These studies suggest that IL-3, but not KL, promotes HPC survival, whereas KL plays a greater role than IL-3 in sustaining more primitive HPC, such as pre-CFC. The effects of both cytokines in mediating HPC and primitive hematopoietic cell survival appear to be related, in part, to their ability to suppress apoptosis.

Constitutive Expression of Steel Factor Gene by Human Stromal Cells

Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

Constitutive expression of steel factor gene by human stromal cells

Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

Cell proliferation and differentiation in chemical leukemogenesis.

In tissues such as bone marrow with normally high rates of cell division, proliferation is tightly coordinated with cell differentiation. Survival, proliferation and differentiation of early hematopoietic progenitor cells depend on the growth factors, interleukin 3 (IL-3) and/or granulocyte-macrophage colony stimulating factor (GM-CSF) and their synergism with other cytokines. We provide evidence that a characteristic shared by a diverse group of compounds with demonstrated leukemogenic potential is the ability to act synergistically with GM-CSF. This results in an increase in recruitment of a resting population of hematopoietic progenitor cells normally unresponsive to the cytokine and a twofold increase in the size of the proliferating cell population normally regarded to be at risk of transformation in leukemogenesis. These findings support the possibility that transient alterations in hematopoietic progenitor cell differentiation may be an important factor in the early stages of development of leukemia secondary to chemical or drug exposure.

Identification of Functionally Distinct Domains of Human Granulocyte-Macrophage Colony-Stimulating Factor Using Monoclonal Antibodies

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40-77, 78-94, or 110-127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.

Identification of functionally distinct domains of human granulocyte- macrophage colony-stimulating factor using monoclonal antibodies

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40–77, 78–94, or 110–127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.

The effect of x-ray dose rate on the clonagenic survival of human granulocyte-macrophage hematopoietic progenitor cells (GM-CFUc) and leukemia cell lines [formula omitted

The effect of x-ray dose rate on the clonagenic survival of human granulocyte-macrophage hematopoietic progenitor cells (GM-CFUc) and leukemia cell lines [formula omitted

Effect of X-ray dose rate on the clonagenic survival of normal human granulocyte-macrophage hematopoietic progenitor cells (Gm-CFUc) in vitro

Effect of X-ray dose rate on the clonagenic survival of normal human granulocyte-macrophage hematopoietic progenitor cells (Gm-CFUc) in vitro