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P M Lansdorp

Publications and source records attributed to P M Lansdorp.

At least 19 recordsLinked to original sources
PubMed1995

Differentiation-associated changes in CD44 isoform expression during normal hematopoiesis and their alteration in chronic myeloid leukemia.

CD44 is a widely expressed, multifunctional, cell-surface glycoprotein that has been implicated in the regulation of normal hematopoiesis. In addition, expression of particular isoforms of CD44 has been associated with malignant transformation and/or the acquisition of metastatic potential. In this study, we used two recently developed monoclonal anti-CD44 antibodies, one reactive with an epitope shared by many CD44 isoforms and the other with an epitope unique to CD44 isoforms containing amino acids encoded by the alternatively spliced exon v10, to compare the expression of CD44 on primitive hematopoietic cells from the marrow of normal individuals and their neoplastic counterparts present in the peripheral blood of patients with chronic myeloid leukemia (CML). Multiparameter fluorescence-activated cell sorter (FACS) analysis and cell sorting studies showed that CD44 is normally expressed at high to very high levels on both long-term culture-initiating cells (LTC-IC) and granulopoietic colony-forming cells (granulocyte-macrophage colony-forming units [CFU-GM]). In contrast, primitive erythropoietic progenitors (burst-forming units-erythroid [BFU-E]) in normal marrow were more homogeneous in their expression of CD44, and very few (less than 5%) showed the very high levels of CD44 seen on 20% to 25% of LTC-IC and CFU-GM. Antibody staining showed the expression of exon v10-containing CD44 isoforms to be restricted to a small subpopulation (4% to 8%) of morphologically recognizable mature (CD34-) myeloid cells within the light-density fraction of normal marrow cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the presence of two exon v10-containing mRNA species. In CML, a significantly greater proportion of the circulating neoplastic CFU-GM expressed very high levels of CD44, and these CFU-GM were accompanied by an increased number of light density v10+ cells, including some that coexpressed CD34. Nonmalignant hematopoietic progenitors mobilized by prior chemotherapy and growth factor treatment of patients with Hodgkin's disease or acute myeloid leukemia in remission showed no changes in CD44 expression relative to normal marrow progenitors. These results provide evidence of early differentiation-associated changes in CD44 expression during normal hematopoiesis in vivo that may be deregulated in the neoplastic clone of patients with CML.

Base Sequence↗
PubMed1995

Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo.

Hox genes were first recognized for their role in embryonic development and may also play important lineage-specific functions in a variety of somatic tissues including the hematopoietic system. We have recently shown that certain members of the Hox A and B clusters, such as HOXB3 and HOXB4, are preferentially expressed in subpopulations of human bone marrow that are highly enriched for the most primitive hematopoietic cell types. To assess the role these genes may play in regulating the proliferation and/or differentiation of such cells, we engineered the overexpression of HOXB4 in murine bone marrow cells by retroviral gene transfer and analyzed subsequent effects on the behavior of various hematopoietic stem and progenitor cell populations both in vitro and in vivo. Serial transplantation studies revealed a greatly enhanced ability of HOXB4-transduced bone marrow cells to regenerate the most primitive hematopoietic stem cell compartment resulting in 50-fold higher numbers of transplantable totipotent hematopoietic stem cells in primary and secondary recipients, compared with serially passaged neo-infected control cells. This heightened expansion in vivo of HOXB4-transduced hematopoietic stem cells was not accompanied by identifiable anomalies in the peripheral blood of these mice. Enhanced proliferation in vitro of day-12 CFU-S and clonogenic progenitors was also documented. These results indicate HOXB4 to be an important regulator of very early but not late hematopoietic cell proliferation and suggest a new approach to the controlled amplification of genetically modified hematopoietic stem cell populations.

Animals↗
PubMed1995

Differential effects of the hematopoietic inhibitors MIP-1 alpha, TGF-beta, and TNF-alpha on cytokine-induced proliferation of subpopulations of CD34+ cells purified from cord blood and fetal liver.

We have previously characterized the proliferative response of primitive CD34+ cells, purified from adult bone marrow, umbilical cord blood, and fetal liver, to a mixture of hematopoietic stimulators (steel factor [SF], interleukin-3 [IL-3], IL-6, and erythropoietin [Epo]) in serum-free liquid cultures. In the present study, we assessed the effects of the hematopoietic inhibitors, macrophage inflammatory protein-1 alpha (MIP-1 alpha), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha), on the cytokine-induced proliferation of three different CD34+ cell subpopulations derived from cord blood and on total CD34+ cells derived from fetal liver. In cultures of cord blood cells, addition of MIP-1 alpha inhibited the numerical expansion of primitive CD34+ cells (CD34+ CD45RAlow CD71low cells) without inhibiting the proliferation of more mature subpopulations enriched for myeloid (CD34+ CD45RA+ CD71low cells) or erythroid (CD34+ CD45RAlow CD71+ cells) progenitors. TGF-beta significantly reduced the proliferation of all three subpopulations, although its effects were more pronounced on cells of the erythroid lineage, particularly immature erythroid progenitors. Similarly, TNF-alpha preferentially inhibited total nucleated and CD34+ cell production in the subpopulation enriched for erythroid cells. However, in contrast to TGF-beta, TNF-alpha preferentially inhibited the proliferation of more mature erythroid progenitors. In a separate set of experiments, MIP-1 alpha, TGF-beta, and TNF-alpha were added to cultures of total CD34+ cells purified from fetal liver. In keeping with the fact that the majority of the progenitors contained in these cells were erythroid progenitors, the inhibitory effects of the three cytokines were similar to those observed in cultures of CD34+ CD45RAlow CD71+ cord blood cells. The results of the present study demonstrate that MIP-1 alpha, TGF-beta, and TNF-alpha have the capacity to modulate cytokine-induced proliferation of cord blood and fetal liver progenitors. The differential effects of these three cytokines confirm their pleiotropic nature as regulators of hematopoiesis.

Antigens, CD↗
PubMed1994

Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells.

Class I homeobox (Hox) genes encode a major group of transcription factors controlling embryonic development and have been implicated in the continuing process of hematopoietic cell differentiation. They are clustered on four chromosomes and, in early development, exhibit spatially restricted expression with respect to their 3'-->5' chromosomal position. By using an improved PCR-based method for amplifying total cDNA derived from limited cell numbers, we now describe the expression of class I Hox genes in highly purified CD34+ cell subpopulations isolated from normal human bone marrow that represent functionally distinct stem and progenitor cell compartments. Our data indicate that at least 16 different Hox genes, mainly from the A and the B clusters, are expressed in one or more of these subpopulations of human hematopoietic cells. Moreover, markedly elevated expression of some of the Hox genes found at the 3' end of the A and B clusters (e.g., HoxB3) was a unique feature of the subpopulations that contained the most primitive functionally defined cells, whereas genes located in the 5' region of each cluster (e.g., HoxA10) were found to be expressed at nearly equal levels in the CD34+ subpopulations analyzed. In contrast to the findings for CD34+ cells, expression of two selected Hox genes, HoxB3 and HoxA10, was virtually extinguished in the CD34- fraction of bone marrow cells. These results demonstrate the expression of a broad range of Hox genes in primitive hematopoietic cells and point to the existence of a regulated program of Hox gene expression during their normal development.

Antigens, CD↗
PubMed1994

Expression of interleukin-1 beta gene in candidate human hematopoietic stem cells.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect interleukin-1 beta (IL-1 beta) mRNA in candidate human hematopoietic stem cells. The cells, obtained from adult bone marrow (BM) or umbilical cord blood, had a CD34+ CD45RAlo CD71lo phenotype and were further fractionated into CD38+ and CD38- or Thy-1+ and Thy-1- subpopulations. The purity of these fractions was always more than 99%. IL-1 beta and CD34 mRNA were detected in pools of 30 BM-derived CD34+ CD45RAlo CD71lo cells. To further exclude any contribution by contaminating cells, individual cells were analyzed for CD34 and IL-1 beta mRNA. Positive results were obtained with 2 of 5 individual BM-derived CD34+ CD45RAlo CD71lo CD38+ cells isolated by micromanipulation after overnight culture in serum-free medium without any exogenous cytokines, and 1 of 10 individual CD34+ CD45RAlo CD71lo CD38- cells isolated immediately after sorting. Moreover, of 10 pools of three BM-derived CD34+ CD45RAlo CD71lo cells cultured overnight in the presence of a mixture of various cytokines (Steel factor, IL-3, IL-6, macrophage colony-stimulating factor [M-CSF], erythropoietin, and IL-3/granulocyte-macrophage colony-stimulating factor [GM-CSF] fusion protein), 5 were positive for IL-1 beta mRNA. This result was compatible with more than 20% (95% confidence limit 0.06-0.61) of the BM cells with the CD34+ CD45RAlo CD71lo phenotype expressing IL-1 beta mRNA. IL-1 beta expression was also consistently observed from day 0 to day 9 in liquid cultures of cord-blood-derived CD34+ CD45RAlo CD71lo Thy-1+ or Thy-1- cells. The cultures contained the same combination of cytokines and resulted in an expansion of cell numbers of up to 400-fold. GM-CSF mRNA was not detected in the equivalent of 75 cells at any day, even though it could be detected with high sensitivity in control stromal cells. Because IL-1 beta is a powerful and pleiotropic biomodulator of cytokines and adhesion molecules, our observations suggest that at least some primitive hematopoietic cells do not merely respond passively to signals from their environment, but may themselves regulate the paracrine production of cytokines from neighboring stromal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1994

Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood.

We have previously shown that the most primitive human hematopoietic cells are included within a cell subpopulation expressing high levels of CD34 and low or undetectable levels of CD45RA and CD71. In this study, cord blood cells with this phenotype were sorted and further separated based on their expression on the Thy-1 antigen. The proliferation and differentiation of the purified cell fractions in response to a mixture of hematopoietic cytokines was analyzed in serum- and stroma-free liquid cultures. Thy-1+ cells (25% of CD34+ CD45RAlo CD71lo cells) were particularly enriched for high proliferative potential colony-forming cells (HPP-CFC; up to 45% of the clonogenic cells), whereas Thy-1- cells were enriched for multipotential colony-forming cells (CFU-MIX; up to 46% of the clonogenic cells). When both subpopulations were cultured in serum-free liquid cultures supplemented with a cytokine mixture that included steel factor, interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein, M-CSF, G-CSF, and erythropoietin, Thy-1+ cells showed a much higher numerical expansion of CD34+ cells (30,000-fold) and colony-forming cells (4,700-fold) than was observed in cultures initiated with Thy-1- cells (900-fold increase in CD34+ cell numbers and 241-fold increase in CFC numbers). Cells coexpressing CD34 and Thy-1 were only transiently expanded (up to 29-fold) and were not detected after day 22 of culture. When CD34+ CD45RAlo CD71lo Thy-1+ cells were cultured, either in semi-solid or liquid cultures, in the presence of anti-Thy-1 antibody, a significant reduction in progenitor cell numbers (particularly HPP-CFC) was observed. In contrast, CD34+ CD45RAlo CD71lo Thy-1- cells were not affected by anti-Thy-1. The results of this study indicate that Thy-1 is expressed on primitive cord blood progenitors with the highest in vitro proliferative potential, and further suggest that Thy-1 is involved in hematopoietic cell development, possibly by mediating a negative signal that results in inhibition of primitive cell proliferation.

Antibodies↗
PubMed1994

Amplification of Sca-1+ Lin- WGA+ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential.

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties, Sca-1 expression (Sca-1+), lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-), and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/- 0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in > or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients, respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor, interleukin-6 (IL-6), and erythropoietin (with or without IL-3), a large increase in total cell number, including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly, this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo, as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum- and stroma cell-free culture, providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype.

Animals↗
PubMed1994

Selective separation of cells using magnetic colloids.

We have developed a reliable, purely immunological means of quantitatively labeling cells with magnetic colloidal dextran iron. Labeled cells can be efficiently separated with a high gradient magnetic filter inside a magnetic field. Separation conditions and filter design can be changed to accommodate large and small scale positive or negative selection. This separation technique has been applied to tumor cell depletions, stem cell enrichment, and large scale preclinical T-cell depletions.

Animals↗
PubMed1994

Age-related decline in proliferative potential of purified stem cell candidates.

Recent studies in our laboratory have shown striking differences in the functional properties of candidate hematopoietic stem cells purified from fetal, neonatal, and adult human tissues. These differences include the ability to produce CD34+ cells, the turnover rate, and the fraction of cells that respond to a mixture of cytokines. All these parameters decrease with the age of the cell donor, and some of these observations are summarized here. Extensive qualitative changes in hematopoietic cells from various stages of development should be taken into account in the design of novel therapeutic strategies.

Adult↗
PubMed1993

Lineage commitment in human hemopoiesis involves asymmetric cell division of multipotent progenitors and does not appear to be influenced by cytokines.

Different models have been proposed to explain lineage commitment in hemopoiesis. Some suggest that lineage commitment occurs in a stochastic manner without the direct influence of extracellular factors; others postulate that cytokines determine whether multipotent cells will become erythroid or granulocyte/macrophage progenitors. In the present study, the patterns of proliferation and differentiation of individually sorted human cord blood-derived primitive hemopoietic cells (highly enriched for multipotent progenitors) were analyzed in a serum-free culture system supplemented with different cytokine combinations. In a first set of experiments, the response of individual cells to different cytokine combinations was compared, whereas in a second set of experiments, single cells were allowed to undergo one division after which the two daughter cells were physically separated and cultured in either the same or different cytokine combinations. Proliferation of progenitor cells was absolutely dependent on cytokines, and the combination of mast cell growth factor plus interleukin 6 was sufficient to induce mitosis. When cytokine combinations favoring erythropoiesis and/or myelopoiesis were added to the cultures, a more vigorous proliferative response of the sorted primitive progenitors was observed. Interestingly, the relative proportions of granulocyte/macrophage, erythroid, and multipotent progenitors remained more or less the same regardless of the cytokine combination used, indicating a permissive rather than an instructive role for cytokines in hemopoietic differentiation. Asymmetric cell divisions, defined as a division that yields two daughter cells with distinct functional properties, were observed in 3-17% of the progenitor cells capable of forming colonies under our experimental conditions. In the rest, symmetric divisions involving multipotent and lineage-committed progenitors were observed. The results of this study demonstrate that the asymmetric cell divisions that occur in the early stages of hemopoiesis at the level of multipotent progenitors cannot be skewed by the addition of specific cytokine combinations. These findings support the hypothesis that lineage commitment in hemopoiesis occurs in a stochastic manner by mechanisms that remain to be elucidated.

Antigens, CD↗
PubMed1993

Positive selection of human blood cells using improved high gradient magnetic separation filters.

High gradient magnetic separators (HGMS) create magnetic field gradients that can be used to attract much smaller and less magnetic particles than those required for conventional magnetic separation techniques. As a result cells can be labeled with submicron magnetic particles and still be separated using an HGMS filter. Typically, HGMS filters consist of random arrays of wire such as stainless steel wool. Wire elements arranged regularly in a filter should allow more efficient separation of cells. Filters were constructed containing ordered wire arrays composed of 430 series stainless steel wire mesh with wire diameters of 50, 100, or 150 microns. The ability of these filters to separate T cells from peripheral blood mononuclear cell suspensions was tested and found superior to random arrays of 302 series stainless steel wire (Thomas et al, 1992). Target cells recognized by OKT5 monoclonal antibody were cross-linked to dextran-iron particles of approximately 20 nm in diameter. Separation conditions were optimized and after one passage through the filter 88% of the OKT5+ cells were recovered in the enriched fraction with 85% purity (%OKT5+). Multiple passages (3 times) could achieve 99% purity with 68% recovery. Variations in separation flow rate had a large effect on the balance between purity and recovery. Optimum separation efficiencies were achieved only when > 10(8) cells were processed. The primarily cause of nonspecific entrapment of CD8- cells was not nonspecific magnetic labeling of cells but the physical (nonmagnetic) characteristics of the filter/filter chamber.

Adult↗
PubMed1993

In vitro properties of purified human stem cell candidates.

Serum-free cultures supplemented with IL-6, IL-3, steel factor, and erythropoietin support extensive production of erythroid cells from purified bone marrow stem cell candidates which are themselves maintained in number in such cultures. In this study, the mechanism responsible for the observed maintenance of primitive hematopoietic cells in rapidly proliferating suspension cultures was examined. The following models were considered: (1) proliferating cells represent a small minority of cells at start of culture (large quiescent pool), (2) self-renewal of primitive cells (balanced by loss through differentiation and death), and (3) asymmetrical divisions (each division of a primitive cell yielding one equally primitive daughter cell and one less primitive, i.e., committed daughter cell). To discriminate between these various models, the proliferative behavior of purified CD34+ CD71 cells was studied at a population level using the fluorescent membrane dye PKH26 and at a single cell level by studying cultures of individually sorted CD34+ CD45RAloCD71lo bone marrow stem cell candidates. The results from these experiments indicate that the majority of purified stem cell candidates do not rapidly proliferate in response to the combination of growth factors used and that limited production of CD34+ cells compensates for the loss of such cells in culture. These observations have to be taken into account in the development of clinical useful strategies for the expansion of primitive hematopoietic bone marrow cells ex vivo.

Antigens, CD↗
PubMed1992

Characterization of primitive hematopoietic cells in normal human peripheral blood.

The total number of clonogenic cells present in 5-week-old long-term cultures (LTC) initiated by seeding normal human marrow cells on competent adherent cell feeder layers allows for the quantitation of a more primitive hematopoietic input precursor cell type referred to as an LTC-initiating cell (LTC-IC). Previous studies have suggested that LTC-IC also circulate because production of clonogenic cells continues for many weeks when cells from the light-density (< 1.077 g/mL), T-cell-depleted fraction of normal blood are maintained on irradiated, marrow-derived feeder layers in LTC medium. We now show that the number of clonogenic cells present in such reconstructed LTC after 5 weeks is linearly related to the input number of peripheral blood (PB) cells over a wide range of cell concentrations, thereby permitting the quantitation of circulating LTC-IC by limiting dilution analysis. Using this approach, we have found the concentration of LTC-IC in the circulation of normal adults to be 2.9 +/- 0.5/mL. This is approximately 75-fold lower than the concentration of circulating clonogenic cells (ie, burst-forming units-erythroid plus colony-forming units [CFU] granulocyte-macrophage plus CFU-granulocyte, erythroid, monocyte, megakaryocyte) and represents a frequency of LTC-IC relative to all nucleated cells that is approximately 100-fold lower than that measured in normal marrow aspirate samples. Characterization studies showed most circulating LTC-IC to be small (low forward light scatter and side scatter), CD34+, Rh-123dull, HLA-DR-, and 4-hydroperoxycyclophosphamide-resistant cells, with differentiative and proliferative potentialities indistinguishable from LTC-IC in normal marrow. Isolation of the light-density, T-cell-depleted, CD34+, and either HLA-DR(low) or Rh-123(dull) fraction of normal blood yielded a highly enriched population of cells that were 0.5% to 1% LTC-IC (approximately 1,500-fold enriched beyond the light-density, T-cell-depletion step), a purity comparable to the most enriched populations of human marrow LTC-IC reported to date. However, purification of PB LTC-IC on the basis of these properties did not allow them to be physically separated from a substantial proportion (> 30%) of the clonogenic cells in the same samples, in contrast to previous findings for LTC-IC and clonogenic cells in marrow. These studies show the presence in the blood of normal adults of a relatively small but readily detectable population of functionally defined, primitive hematopoietic cells that share properties with marrow LTC-IC, a cell type thought to have in vivo reconstituting potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1992

Phenotypic heterogeneity of primitive leukemic hematopoietic cells in patients with chronic myeloid leukemia.

The peripheral blood of chronic myeloid leukemia (CML) patients with chronic-phase disease and elevated white blood cell (WBC) counts typically contains markedly increased numbers of a variety of neoplastic pluripotent and lineage-restricted hematopoietic progenitors. These include cells detected in standard colony assays as well as their more primitive precursors. The latter are referred to as long-term culture-initiating cells (LTC-IC) because of their ability to generate clonogenic cell progeny detectable after a minimum of 5 weeks incubation on competent fibroblast feeder layers. In this study, we have investigated a number of the properties of the LTC-IC and clonogenic cells present in the blood of such CML patients with high WBC counts. This included an analysis of the light scattering properties of these progenitors, as well as their expression of CD34 and HLA-DR, Rhodamine-123 staining, and in vitro sensitivity to 4-hydroperoxycyclophosphamide. In the case of LTC-IC, the production of different types of lineage-restricted and multipotent progeny was also analyzed. Most of the circulating LTC-IC and clonogenic cells in the CML patients studied (on average approximately 70% and approximately 90%, respectively) showed features of proliferating or activated cells. This is in marked contrast to the majority of progenitors in the blood of normal individuals and most of the LTC-IC in normal marrow, all of which exhibit a phenotype expected of quiescent cells. Interestingly, a significant proportion of the circulating clonogenic cells and LTC-IC in the CML samples studied (on average approximately 10% and approximately 30%, respectively) appeared to be phenotypically similar to normal circulating progenitors, although their absolute numbers were indicative of a neoplastic origin. Both phenotypes of circulating CML clonogenic cells and LTC-IC could be obtained at approximately 10% to 20% purity by differential multiparameter sorting. These findings suggest that expansion of the Philadelphia chromosome-positive clone at the level of the earliest types of hematopoietic cells results from the activation of mechanisms that enable some, but not all, signals that block the cycling of normal stem cells to be bypassed or overcome. In addition, they provide strategies for purifying these primitive leukemic cells that should facilitate further analysis of the mechanisms underlying their abnormal proliferative behavior.

Antigens, CD↗
PubMed1992

High gradient magnetic separation of cells on the basis of expression levels of cell surface antigens.

The possibility of separating cells on the basis of levels of antigen expression was explored in a model system using fixed erythrocytes and high gradient magnetic separation (HGMS). Fixed human erythrocytes were labelled to varying degrees with tetrameric monoclonal antibody complexes specific for both dextran and glycophorin A-M. The cells were then mixed and incubated with dextran iron particles prior to magnetic separation. The small size of the dextran iron particles (less than 0.2 microns) resulted in quantitative magnetic labelling of cells as shown using fluoresceinated anti-dextran antibodies and flow cytometry. The relationships between the initial percentage of labelled cells, cell recovery, non-specific entrapment of unlabelled cells, the purity of the removed fraction, the degree of antigen expression and separation conditions (flow rate and field strength) were determined and used to establish separation conditions that allowed recovery of cells that differ only in the degree of antibody labelling.

Antibodies, Monoclonal↗
PubMed1992

Long-term erythropoiesis from constant numbers of CD34+ cells in serum-free cultures initiated with highly purified progenitor cells from human bone marrow.

To directly study the biological properties of purified hematopoietic colony-forming cell precursors, cells with a CD34+ CD45RAlo CD71lo phenotype were purified from human bone marrow using density separation and fluorescence-activated cell sorting, and were cultured in serum-free culture medium supplemented with various cytokines. In the presence of interleukin 3 (IL-3), IL-6, erythropoietin, and mast cell growth factor (a c-kit ligand), cell numbers increased approximately 10(6)-fold over a period of 4 wk, and the percentage of cells that expressed transferrin receptors (CD71) increased from less than 0.1% at day 0 to greater than 99% at day 14. Interestingly, the absolute number of CD34+ CD71lo cells did not change during culture. When CD34+ CD71lo cells were sorted from expanded cultures and recultured, extensive cell production was repeated, again without significant changes in the absolute number of cells with the CD34+ CD71lo phenotype that were used to initiate the (sub)cultures. These results document that primitive hematopoietic cells can generate progeny without an apparent decrease in the size of a precursor cell pool.

Antigens, CD↗
PubMed1992

Increased erythropoietin-receptor expression on CD34-positive bone marrow cells from patients with chronic myeloid leukemia.

Erythropoietin-receptor (EpR) expression on bone marrow cells from normal individuals and from patients with chronic myeloid leukemia (CML) was examined by multiparameter flow cytometry after stepwise amplified immunostaining with biotin-labeled Ep, streptavidin-conjugated R-phycoerythrin, and biotinylated monoclonal anti-R-phycoerythrin. This approach allowed the detection of EpR-positive cells in all bone marrow samples studied. Most of the EpR-positive cells in normal bone marrow were found to be CD45-dull, CD34-negative, transferrin-receptor-positive and glycophorin-A-intermediate to -positive. This phenotype is characteristic of relatively mature erythroid precursors, ie, colony-forming units-erythroid and erythroblasts recognizable by classic staining procedures. Approximately 5% of normal EpR-positive cells displayed an intermediate expression of CD45, suggesting that these represented precursors of the CD45-dull EpR-positive cells. Some EpR-positive cells in chronic myeloid leukemia (CML) bone marrow had a phenotype similar to the major EpR-positive phenotype in normal bone marrow, ie, CD34-negative and CD45-dull. However, there was a disproportionate increase in the relative number of EpR-positive/CD45-intermediate cells in CML bone marrow. Even more striking differences between normal individuals and CML patients were observed when EpR-expression on CD34-positive marrow cells was analyzed. Very few EpR-positive cells were found in the CD34-positive fraction of normal bone marrow, whereas a significant fraction of the CD34-positive marrow cells from five of five CML patients expressed readily detectable EpR. These findings suggest that control of EpR expression is perturbed in the neoplastic clone of cells present in patients with CML. This may be related to the inadequate output of mature red blood cells typical of CML patients and may also be part of a more generalized perturbation in expression and/or functional integrity of other growth factor receptors on CML cells.

Antigens, CD↗