Pluripotent Hematopoietic Cells Research Articles

Enforced Expression of the GATA-2 Transcription Factor Blocks Normal Hematopoiesis

AbstractThe zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1+Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1+ Lin−cells. Although Sca-1+ Lin− bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1+Lin− cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

Enforced Expression of the GATA-2 Transcription Factor Blocks Normal Hematopoiesis

The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1+Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1+ Lin−cells. Although Sca-1+ Lin− bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1+Lin− cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

Protein Kinase C Mediates the Mitogenic Action of Thrombopoietin in c-Mpl–Expressing UT-7 Cells

AbstractProtein kinase C (PKC) has been implicated in signal transduction events elicited by several hematopoietic growth factors. Thrombopoietin (TPO) is the major regulator of megakaryocytic lineage development, and its receptor, c-Mpl, transduces signals for the proliferation and differentiation of hematopoietic progenitors. In this study we have examined the effect of TPO on the subcellular distribution of PKC (a measure of enzyme activation) in a growth factor-dependent pluripotent hematopoietic cell line that was engineered to express the c-Mpl receptor (UT-7/mpl). In addition, we have assessed the significance of this activation for the induction of both mitogenesis and differentiation. Using a PKC translocation assay, TPO was found to stimulate a time- and dose-dependent increase in the total content of PKC activity present in the membrane fraction of UT-7/mpl cells (maximum increase = 2.3-fold above basal level after 15 minutes with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that TPO treatment led to a marked increase of the Ca2+/diacylglycerol-sensitive PKC isoforms α and β found in the membrane fraction. In contrast, the subcellular distribution of these isoforms did not change after treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of UT-7/mpl cells to the selective PKC inhibitor GF109203X completely inhibited the PKC activity associated to the membrane fraction after TPO treatment, and blocked the mitogenic effect of TPO. In contrast, GF109203X had no effect on the TPO-induced expression of GpIIb, a megakaryocytic differentiation antigen. Downregulation of PKC isoforms α and β to less than 25% of their initial level by treatment with phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic response, but had no significant effect when this response was induced by GM-CSF. Taken together, these findings suggest that (1) TPO stimulates the activation of PKC, (2) PKC activation mediates the mitogenic action of TPO, and (3) PKC activation is not required for TPO-induced expression of megakaryocytic surface markers.

Protein Kinase C Mediates the Mitogenic Action of Thrombopoietin in c-Mpl–Expressing UT-7 Cells

Protein kinase C (PKC) has been implicated in signal transduction events elicited by several hematopoietic growth factors. Thrombopoietin (TPO) is the major regulator of megakaryocytic lineage development, and its receptor, c-Mpl, transduces signals for the proliferation and differentiation of hematopoietic progenitors. In this study we have examined the effect of TPO on the subcellular distribution of PKC (a measure of enzyme activation) in a growth factor-dependent pluripotent hematopoietic cell line that was engineered to express the c-Mpl receptor (UT-7/mpl). In addition, we have assessed the significance of this activation for the induction of both mitogenesis and differentiation. Using a PKC translocation assay, TPO was found to stimulate a time- and dose-dependent increase in the total content of PKC activity present in the membrane fraction of UT-7/mpl cells (maximum increase = 2.3-fold above basal level after 15 minutes with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that TPO treatment led to a marked increase of the Ca2+/diacylglycerol-sensitive PKC isoforms α and β found in the membrane fraction. In contrast, the subcellular distribution of these isoforms did not change after treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of UT-7/mpl cells to the selective PKC inhibitor GF109203X completely inhibited the PKC activity associated to the membrane fraction after TPO treatment, and blocked the mitogenic effect of TPO. In contrast, GF109203X had no effect on the TPO-induced expression of GpIIb, a megakaryocytic differentiation antigen. Downregulation of PKC isoforms α and β to less than 25% of their initial level by treatment with phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic response, but had no significant effect when this response was induced by GM-CSF. Taken together, these findings suggest that (1) TPO stimulates the activation of PKC, (2) PKC activation mediates the mitogenic action of TPO, and (3) PKC activation is not required for TPO-induced expression of megakaryocytic surface markers.

Immune phenotype of canine hematopoietic progenitor cells.

The immune phenotype of canine hematopoietic progenitor cells was studied by immunoseparation and culturing of separated cells. Two separation methods were used, the magnetic cell sorting system (MACS) and the fluorescence activated cell sorter (FACS). For separation rat anti dog antibodies Dog 13 and Dog 14 directed against Thy-1, and Dog 26 as well as cross-reactive mouse anti human antibodies IOT2a and 7.2 directed against MHC class II were used. Separated cell populations were cultured in semisolid agar before and after long-term culture on a pre-established irradiated stromal cell layer. After 28 days, adherent and nonadherent cells were harvested from long-term culture. The MACS system allowed separation of cells into positive and negative fractions. Long-term culture-initiating cells (LTC-IC) were found in both the Thy-1+ and the Thy-1- fraction, but the content of LTC-IC was higher in the Thy-1+ fraction. The MACS system did not allow separation of progenitor cells according to the expression of MHC class II antigen detected by Dog 26 and the cross-reactive antibodies IOT2a and 7.2. In contrast to the MACS system the FACS allowed separation of negative, low-positive and high-positive cell populations. Low-positive fractions were well defined for Thy-1 and less well defined for MHC class II. CFU before and after long-term culture were exclusively observed in the low positive fraction (Thy-1(lo+)). Using MHC class II antibody Dog 26 LTC-IC were found mainly in the negative and low positive fraction, and CFU were observed mainly in the low and high positive fraction. In conclusion pluripotent canine hematopoietic precursor cells are low positive for Thy-1 and for MHC class II. In this respect canine hematopoietic progenitor cells are comparable to those of mouse and man.

Molecular Cloning and Characterization of Lysosomal Sialic Acid O-Acetylesterase

O-Acetylation and de-O-acetylation of sialic acids have been implicated in the regulation of a variety of biological phenomena, including endogenous lectin recognition, tumor antigenicity, virus binding, and complement activation. Applying a strategy designed to identify genes preferentially expressed in active sites of embryonic hematopoiesis, we isolated a novel cDNA from the pluripotent hematopoietic cell line FDCPmixA4 whose open reading frame contained sequences homologous to peptide fragments of a lysosomal sialic acid O-acetylesterase (Lse) previously purified from rat liver, but with no evident similarity to endoplasmic reticulum-derived acetylesterases. The expressed Lse protein exhibits sialic-acid O-acetylesterase activity that is not attributable to a typical serine esterase active site. lse expression is spatially and temporally restricted during embryogenesis, and its mRNA levels correlate with differences in O-acetylesterase activity described in adult tissues and blood cell types. Using interspecific backcross analysis, we further mapped the lse gene to the central region of mouse chromosome 9. This constitutes the first report on the molecular cloning of a sialic acid-specific O-acetylesterase in vertebrates and suggests novel roles for the 9-O-acetyl modification of sialic acids during the development and differentiation of mammalian organisms.

BCR-ABL Does Not Prevent Apoptotic Death Induced by Human Natural Killer or Lymphokine-Activated Killer Cells

The erythromyeloid cell line, K562, the most sensitive target in human natural killer (NK) cell mediated cytotoxicity, is derived from a chronic myeloid leukemia (CML) patient and expresses the characteristic reciprocal translocation t(9;22). The resulting BCR-ABL fusion protein has been shown to mediate the unusual resistance of K562, and other BCR-ABL expressing lines, to apoptosis induced by a variety of agents (irradiation, UV light, cytotoxic drugs). Here we show that human NK and lymphokine-activated killer (LAK) cells, when tested at low effector to target ratio, can readily induce apoptotic death in K562 cells. This was accompanied with classical DNA oligonucleosomal fragmentation, an unexpected finding given the reported lack of such fragmentation when apoptosis is induced in K562 by chemical agents, after downregulation of BCR-ABL. Apoptosis was assessed by several means: morphological studies, 125I-DNA versus 51Cr release, DNA agarose gel electrophoresis, and results were always concordant, with a delayed kinetics for DNA oligonucleosomal fragmentation. Similar data were obtained with a pluripotent human hematopoietic cell line, UT-7, infected with a defective amphotropic p210 BCR-ABL retrovirus. The BCR-ABL expressing subclone UT-7/9, while being no longer sensitive to cytotoxic drugs or to tumor necrosis factor, a lytic mediator to which UT-7 cells are sensitive, underwent apoptotic death when exposed to LAK effector cells to the same degree as the parental UT-7 line. With these targets, DNA oligonucleosomal fragmentation occurred concomitantly with isotope release. Results obtained with several inhibitors of exocytosis strongly suggest that cytotoxic granules mediate NK and LAK cell-induced apoptotic death. In conclusion, NK and LAK cell-induced apoptotic signals, unlike those activated by chemotherapeutic agents, are unaffected by the antiapoptotic action of BCR-ABL. This unique property may support the observed curative effect of allogeneic bone marrow transplantation in CML.

Characterization of hematopoietic cells arising on the textured surface of left ventricular assist devices

Textured biomaterial surfaces in implantable left ventricular assist devices induce development of a nonthrombotic neointimal surface and allow elimination of anticoagulation therapy in device recipients. Characterization of the hematopoietic cells formed within the neointimal surfaces of these devices will contribute to our understanding of this unique neointima. The blood-contacting surface of seven ThermoCardiosystems left ventricular assist devices was removed, washed with phosphate-buffered saline solution, and digested with 0.1% collagenase for 15 to 20 minutes. The hematopoietic cells released from the explants were isolated and analyzed by flow cytometry and immuno-histochemical staining. More than 80% +/- 6% of hematopoietic cells isolated in this fashion are of myelomonocytic origin and express CD14, CD15, and CD33 surface molecules. Four percent of cells express the CD34 surface marker, which suggests that the neointima is colonized by pluripotent hematopoietic stem cells. Continuous culture of these hematopoietic cells in the presence of the cytokines interleukin-3, c-kit ligand, granulocyte colony-stimulating factor resulted in tenfold expansion by day 7 and 25-fold expansion by day 14. Pluripotent hematopoietic cells with a high proliferative capacity colonize textured surfaces of left ventricular assist devices and may contribute to the development of a biologically nonthrombogenic neointima.

Regulation of alpha 2 integrin gene expression in cells with megakaryocytic features: a common theme of three necessary elements

The alpha 2 beta 1 integrin mediates interactions between cells and the extracellular matrix molecules, collagen and/or laminin. The alpha 2 beta 1 integrin is expressed in a variety of cell types, but in cells of hematopoietic lineage, expression is restricted to megakaryocytes and platelets. Increased expression of the alpha 2 beta 1 integrin during megakaryocytic differentiation is a consequence of transcriptional activation of the alpha 2 gene. We have begun to characterize the role of the 52 flanking region of the alpha 2 integrin gene in regulating expression during megakaryocyte differentiation. A 5-kb fragment of the 52 region directs both cell type and differentiation-dependent expression of a reporter gene in the pluripotent hematopoietic K562 cells upon megakaryocytic differentiation and in the megakaryocytic cell line, Dami. Analysis of a series of 52 deletion mutants indicates that expression of the alpha 2 integrin gene in cells with megakaryocytic features requires a core promoter region, a silencer region, and megakaryocytic enhancers in the distal 52 end. The organization of these three distinct regulatory regions of the alpha 2 promoter/enhancer suggests a common theme for megakaryocytic gene regulation shared with other megakaryocyte-specific proteins, including alpha IIb integrin subunit and platelet factor 4.

Regulation of α2 Integrin Gene Expression in Cells With Megakaryocytic Features: A Common Theme of Three Necessary Elements

The alpha 2 beta 1 integrin mediates interactions between cells and the extracellular matrix molecules, collagen and/or laminin. The alpha 2 beta 1 integrin is expressed in a variety of cell types, but in cells of hematopoietic lineage, expression is restricted to megakaryocytes and platelets. Increased expression of the alpha 2 beta 1 integrin during megakaryocytic differentiation is a consequence of transcriptional activation of the alpha 2 gene. We have begun to characterize the role of the 5' flanking region of the alpha 2 integrin gene in regulating expression during megakaryocyte differentiation. A 5-kb fragment of the 5' region directs both cell type and differentiation-dependent expression of a reporter gene in the pluripotent hematopoietic K562 cells upon megakaryocytic differentiation and in the megakaryocytic cell line, Dami. Analysis of a series of 5' deletion mutants indicates that expression of the alpha 2 integrin gene in cells with megakaryocytic features requires a core promoter region, a silencer region, and megakaryocytic enhancers in the distal 5' end. The organization of these three distinct regulatory regions of the alpha 2 promoter/enhancer suggests a common theme for megakaryocytic gene regulation shared with other megakaryocyte-specific proteins, including alpha IIb integrin subunit and platelet factor 4.

The human alpha 2 integrin gene promoter. Identification of positive and negative regulatory elements important for cell-type and developmentally restricted gene expression.

The alpha 2 beta 1 serves as a collagen receptor or a collagen/laminin receptor, depending upon cell type. Expression of the integrin is regulated during normal cellular differentiation and is altered during carcinogenesis. We have previously demonstrated that increased expression of the alpha 2 beta 1 integrin during megakaryocytic differentiation is a consequence of increased alpha 2 mRNA due to transcriptional activation of the alpha 2 integrin gene and that the decreased expression of the integrin in breast adenocarcinoma is due to decreased steady-state levels of alpha 2 mRNA. We now report the identification and characterization of the 5'-flanking region of the alpha 2 integrin gene. The 5'-untranslated region of the alpha 2 mRNA extends 129 base pairs 5' to the site of translation initiation. The promoter region lacks TATA and CAAT boxes but contains an abbreviated initiator sequence and six Sp1 binding sites. Consensus binding sites for AP-1 and AP-2 complexes, a GATA box, a Pu.1 box, and two palindromic motifs with potential to bind the estrogen receptor are also present. A 961-base pair fragment of the 5'-flanking region directs both cell type- and differentiation-specific expression of a reporter gene in T47-D epithelial cells and in pluripotent hematopoietic K562 cells upon megakaryocytic differentiation.

Expression of human colony-stimulating factor-1 (CSF-1) receptor in murine pluripotent hematopoietic NFS-60 cells induces long-term proliferation in response to CSF-1 without loss of erythroid differentiation potential

NFS-60 and FDCP-Mix cells are interleukin-3--dependent multipotent hematopoietic cells that can differentiate in vitro into mature myeloid and erythroid cells. Retrovirus-mediated transfer of the human colony- stimulating factor-1 (CSF-1) receptor gene (c-fms) enabled NFS-60 cells but not FDCP-Mix cells to proliferate in response to CSF-1. The phenotype of NFS-60 cells expressing the human CSF-1 receptor (CSF-1R) grown in CSF-1 did not grossly differ from that of original NFS-60 as assessed by cytochemical and surface markers. Importantly, these cells retained their erythroid potentiality. In contrast, a CSF-1-dependent variant of NFS-60, strongly expressing murine CSF-1R, differentiated into monocyte/macrophages upon CSF-1 stimulation and almost totally lost its erythroid potentiality. We also observed that NFS-60 but not FDCP-Mix cells could grow in response to stem cell factor, (SCF), although both cell lines express relatively high amounts of SCF receptors. This suggests that SCF-R and CSF-1R signalling pathways share at least one component that may be missing or insufficiently expressed in FDCP-Mix cells. Taken together, these results suggest that human CSF-1R can use the SCF-R signalling pathway in murine multipotent cells and thereby favor self-renewal versus differentiation.

Expression of Human Colony-Stimulating Factor-1 (CSF-1) Receptor in Murine Pluripotent Hematopoietic NFS-60 Cells Induces Long-Term Proliferation in Response to CSF-1 Without Loss of Erythroid Differentiation Potential

AbstractNFS-60 and FDCP-Mix cells are interleukin-3–dependent multipotent hematopoietic cells that can differentiate in vitro into mature myeloid and erythroid cells. Retrovirus-mediated transfer of the human colony-stimulating factor-1 (CSF-1) receptor gene (c-fms) enabled NFS-60 cells but not FDCP-Mix cells to proliferate in response to CSF-1. The phenotype of NFS-60 cells expressing the human CSF-1 receptor (CSF-1R) grown in CSF-1 did not grossly differ from that of original NFS-60 as assessed by cytochemical and surface markers. Importantly, these cells retained their erythroid potentiality. In contrast, a CSF-1–dependent variant of NFS-60, strongly expressing murine CSF-1 R, differentiated into monocyte/macrophages upon CSF-1 stimulation and almost totally lost its erythroid potentiality. We also observed that NFS-60 but not FDCP-Mix cells could grow in response to stem cell factor, (SCF), although both cell lines express relatively high amounts of SCF receptors. This suggests that SCF-R and CSF-1 R signalling pathways share at least one component that may be missing or insufficiently expressed in FDCP-Mix cells. Taken together, these results suggest that human CSF-1 R can use the SCF-R signalling pathway in murine multipotent cells and thereby favor self-renewal versus differentiation.

Transcriptional regulation of a hematopoietic proteoglycan core protein gene during hematopoiesis.

The expression of a hematopoietic proteoglycan core protein (HpPG) gene is up-regulated during the early stages of myeloblast differentiation at a time point coinciding with the beginning of granule genesis (Stellrecht, C. M., Mars, W. M., Miwa, H., Beran, M., and Saunders, G. F. (1991) Differentiation 48, 127-135). The mechanism of this up-regulatory event was investigated by analyzing the expression and regulation of the HpPG gene during the differentiation of the pluripotent hematopoietic cell line, K562. The level of HpPG gene expression in these cells was up-regulated approximately 10-fold upon 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation, as measured by Northern blot analysis. The HpPG gene's expression remained relatively unchanged during hemin-induced erythroid differentiation, further demonstrating the specificity of this regulatory event for granule-producing cell lineages. The effect of TPA induction on HpPG gene expression was also assessed during the differentiation of the myeloid leukemia cell line, HL-60. The expression of the gene was down-regulated approximately 20-fold upon TPA-induced differentiation into macrophage-like cells. In contrast, only a minimal decrease in HpPG gene expression was detected in gamma-interferon-induced monocyte differentiation. No detectable changes in expression levels were seen in HL-60 cells differentiated into granulocytes with retinoic acid or dimethyl sulfoxide. Nuclear runoff analysis demonstrated that the regulation of the HpPG gene is under transcriptional control in both TPA-induced differentiation systems.

Substances from the medicinal mushroom Daedalea gibbosa inhibit kinase activity of native and T315I mutated Bcr-Abl

Human chronic myelogenous leukemia (CML) is a malignancy of pluripotent hematopoietic cells characterized by a distinctive cytogenetic abnormality resulting in the creation of a p210 Bcr-Abl fusion protein with abnormal tyrosine kinase activity. Recently, a selective Abl kinase inhibitor, Imatinib mesylate, was introduced as a first line therapy for CML. Despite the initial response, CML patients develop a resistantance to Imatinib, which is mediated mainly by point mutations within the Abl protein. Herein, we describe the identification of mycelium organic extracts of Daedalea gibbosa with selective anti-proliferating and apoptosis-inducing activities against K562 cells and other laboratory model of CML. Using activity-guided purification, we isolated an active fraction, F6, which inhibits in vitro kinase activity of recombinant Abl. The active fraction significantly inhibits the autophosphorylation of native and mutated Bcr-Abl, which are resistant to Imatinib treatment including the T315I mutation. Using a colony-forming assay, we demonstrated that the active fraction is effective in inhibiting the colony formation of the Ba/F3 cell line harboring either native Bcr-Abl or its mutations, including the T315I mutation. Our data illustrated the potential of natural products in cancer therapeutics.

Kinetics of the Appearance of Fc€RI-Bearing Cells in Interleukin-3-Dependent Mouse Bone Marrow Cultures: Correlation With Histamine Content and Mast Cell Maturation

While it is known that mast cells arise from pluripotential hematopoietic cells and express their mature phenotypes in tissues, the sequence of events in maturation is incompletely understood. To study early mast cells, we sorted cells from interleukin-3 (IL-3)-dependent mouse bone marrow cultures on the basis of Fc epsilon RI and examined their morphology, histamine content, and growth characteristics. Flow cytometric analysis and sort showed that the Fc epsilon RI-bearing (Fc epsilon RI+) cells increased from 0% on day 0 to 90% by day 21 and that the total number of Fc epsilon RI+ cells increased from 0 at the start of culture to 3.75 x 10(5) cells by day 21 from an initial population of 1 x 10(5) cells. The dissociation rate of 125I-labeled IgE from early cultured cells resembled the dissociation rate of mouse IgE from mature murine mast cells. Mean fluorescence intensity increased over time, reflecting an increase in IgE receptor density. Fc epsilon RI+ cells were also positive for Fc gamma RII/III. Morphologic studies showed gradual acquisition of metachromatic granules in the Fc epsilon RI+ cells, which was paralleled by an increase in histamine content. Sorted Fc epsilon RI+ cells, when placed in liquid suspension culture, gave rise to pure mast cell populations. Fc epsilon RI+ cells sorted at day 3 and cultured in agarose with IL-3 gave rise to 4,800 small and 150 medium-size mast cell colony-forming units per 10(6) cells, while Fc epsilon RI- cells gave rise to 23 medium-size and 49 large mast cell colony-forming units per 10(6) cells. Fc epsilon RI+ cells grown in granulocyte-macrophage colony-stimulating factor (CSF) or macrophage-CSF did not give rise to colony-forming units. These results show that Fc epsilon RI+ cells have proliferative potential, but that there also is a population of mast cell progenitor cells that have not yet expressed Fc epsilon RI, and such individual progenitor cells have greater potential for proliferation than cells that express Fc epsilon RI.

Kinetics of the appearance of Fc epsilon RI-bearing cells in interleukin-3-dependent mouse bone marrow cultures: correlation with histamine content and mast cell maturation

While it is known that mast cells arise from pluripotential hematopoietic cells and express their mature phenotypes in tissues, the sequence of events in maturation is incompletely understood. To study early mast cells, we sorted cells from interleukin-3 (IL-3)-dependent mouse bone marrow cultures on the basis of Fc epsilon RI and examined their morphology, histamine content, and growth characteristics. Flow cytometric analysis and sort showed that the Fc epsilon RI-bearing (Fc epsilon RI+) cells increased from 0% on day 0 to 90% by day 21 and that the total number of Fc epsilon RI+ cells increased from 0 at the start of culture to 3.75 x 10(5) cells by day 21 from an initial population of 1 x 10(5) cells. The dissociation rate of 125I-labeled IgE from early cultured cells resembled the dissociation rate of mouse IgE from mature murine mast cells. Mean fluorescence intensity increased over time, reflecting an increase in IgE receptor density. Fc epsilon RI+ cells were also positive for Fc gamma RII/III. Morphologic studies showed gradual acquisition of metachromatic granules in the Fc epsilon RI+ cells, which was paralleled by an increase in histamine content. Sorted Fc epsilon RI+ cells, when placed in liquid suspension culture, gave rise to pure mast cell populations. Fc epsilon RI+ cells sorted at day 3 and cultured in agarose with IL-3 gave rise to 4,800 small and 150 medium-size mast cell colony-forming units per 10(6) cells, while Fc epsilon RI- cells gave rise to 23 medium-size and 49 large mast cell colony-forming units per 10(6) cells. Fc epsilon RI+ cells grown in granulocyte-macrophage colony-stimulating factor (CSF) or macrophage- CSF did not give rise to colony-forming units. These results show that Fc epsilon RI+ cells have proliferative potential, but that there also is a population of mast cell progenitor cells that have not yet expressed Fc epsilon RI, and such individual progenitor cells have greater potential for proliferation than cells that express Fc epsilon RI.

CD7+, CD4−/CD8− acute leukemia with t(11;14)(p15;q11) in a child

A t(11;14)(p15;q11) was the sole chromosome abnormality observed in the malignant cells of a 10-year-old boy with acute leukemia. Morphologically, these cells were classified as L1 by the criteria of the French-American-British Working Group. Cytochemical analysis revealed that the leukemic cells were negative for Sudan Black B, periodic acid Schiff, and esterases, and positive for acid phosphatase. Immunophenotyping disclosed that the cells expressed a very immature antigenic profile [CD34+, CD7+, cytoplasmic CD3+, membrane CD3−, CD4−, and CD8−]. In spite of very intensive chemotherapy, complete remission was never induced, and the child died of progressive disease. The relationship of this case to other reported cases of acute leukemia arising from immature pluripotent hematopoietic cells is discussed.

Effect of intensified chemotherapy on the pluripotent haematopoietic progenitor cells CFU-GEMM in adult acute lymphoblastic leukaemia.

In order to analyse the short-term and long-term effects of intensified induction chemotherapy, the frequency, cellular composition and proliferative state of the pluripotent haemopoietic progenitor cells CFU-GEMM was investigated in a total of 35 patients with acute lymphoblastic or acute undifferentiated leukaemia at diagnosis, as well as during and after therapy. At diagnosis, the number of CFU-GEMM/ml bone marrow aspirate was significantly reduced to 12.1 (95% confidence interval 2.1-70) as compared to healthy controls (50.4/ml; 95% confidence interval 66-3846/ml). While immediately after the end of induction therapy, the respective values for CFU-GEMM were still decreased (30.4/ml; 95% confidence interval 8.4-108); values not significantly different from normal were reached within the following 4 weeks. During and after cessation of maintenance therapy again no significant deviation from normal values was observed. Immunological analysis of single colonies revealed that during the early phase after remission induction only 60 +/- 7% of mixed colonies contained megakaryocytic cells (normal 86 +/- 3%; P less than 0.01) while normal values were found during and after maintenance therapy. Cell cycle analysis disclosed highly increased proliferative activity of pluripotent progenitor cells during marrow regeneration after induction therapy as well as during maintenance therapy, but a return to the resting state after cessation of maintenance therapy. It is concluded that this intensive chemotherapy regimen does not result in any apparent long-term damage to the pluripotent haemopoietic progenitor cells.

Effect of cytotoxic lymphocytes obtained by in vitro immunization with syngeneic lymphoma cells on pluripotent hematopoietic cells

The possibility of the presence of leukemia-associated antigens on pluripotent hemopoietic cells was studied with the aid of immune lymphocytes, cytotoxic against mouse syngeneic lymphoma cells. Cytotoxic lymphocytes were obtained during immunization in vitro of C57BL/6 mouse splenocytes by syngeneic T lymphoma EL-4 cells in the presence of interleukin-2. Specific cytotoxic activity of immune lymphocytes as regards EL-4 cells was not blocked by addition of normal bone marrow cells. Incubation of the bone marrow with immune killers did not lead to a decrease in the number of colony-forming units in the spleen. It was shown that using cytotoxic lymphocytes the total killing of lymphoma cells might be achieved in a mixture of bone marrow and lymphoma cells, whereas pluripotent precursor cells might be retained.