Coexpression of Kit and the receptors for erythropoietin, interleukin 6 and GM-CSF on hemopoietic cells.

The detection of functional growth factor (GF) receptors on subpopulations of hemopoietic cells may provide a further dissection of immature cell subsets. Since little information is available about coexpression of different GF receptors at the level of single hemopoietic cells, we studied the feasibility of simultaneous cell staining with a combination of biotin- and digoxigenin-labeled GFs for flow cytometric detection of functional receptors. Using this methodology, coexpression of Kit and receptors for erythropoietin (EPO), interleukin 6 (IL-6), and GM-CSF on hemopoietic cells was studied by triple-staining of rhesus monkey bone marrow (BM) cells with labeled GFs and antibodies against other cell surface markers. Most of the immature, CD34+2 cells were Kit+ but did not display detectable levels of EPO-receptors (EPO-Rs) or GM-CSF-R. Approximately 60% of these CD34+2/Kit+ cells coexpressed the IL-6-R, demonstrating that immature cells are heterogeneous with respect to IL-6-R expression. Maturation of monomyeloid progenitors, as demonstrated by decreasing CD34 and increasing CD11b expression, is accompanied by a decline of Kit and an increase in GM-CSF-R expression in such a way that Kit+/GM-CSF-R+ cells are hardly detectable. IL-6-R expression is maintained or even increased during monomyeloid differentiation. IL-6-R and GM-CSF-R were not identified on most CD71+2 cells, which indicated that these receptors are probably not expressed during erythroid differentiation. Together with previous results, our data show that both Kit and CD71 are upregulated with erythroid commitment of immature progenitors. Upon further differentiation, Kit+/EPO-R-cells lose CD34 and acquire EPO-R. Maturing erythroid cells eventually lose CD71 and Kit expression but retain the EPO-R. In conclusion, this approach enables further characterization of the specificity of GFs for different bone marrow subpopulations. Apart from insight into the differentiation stages on which individual GFs may act, information about receptor coexpression may be used to identify individual cells that can respond to multiple GFs, and allows for further characterization of the regulation of lineage-specific differentiation.

Differential expression of receptors for hemopoietic growth factors on subsets of CD34+ hemopoietic cells.

The production of peripheral blood cells is regulated by hemopoietic growth factors (HGF) which promote the survival of stem cells and stimulate the proliferation and maturation of progenitors as well as effector functions of mature blood cell subsets. The actions of HGF's are determined by the cellular distribution of receptors for these HGF's within the hemopoietic tissues and by the functional program that receptor-expressing cells can execute after growth factor stimulation. Identification of stem cells and their progeny and delineation of the growth factor receptor phenotype of these cells will establish target cell range and functions of individual growth factors in hemopoiesis. Cells with specific HGF receptors can be detected and isolated by flow cytometric methods, e.g., by staining with biotinylated ligand and fluorescently-tagged streptavidin. Receptor-expressing cells can be classified on the basis of expression of the CD34 antigen and other markers that distinguish immature progenitors from more differentiated cells. Using this approach distinct expression patterns have been shown for the receptors for interleukin-3 (IL-3), IL-6, granulocyte/macrophage colony-stimulating factor (GM-CSF) and Steel Factor (SF) on subsets of CD34+ and CD34- cells in bone marrow. Expression of the IL-3 receptor (R), IL-6R and GM-CSFR appears to be very low on the most immature subsets of CD34+ cells, but increases progressively during successive stages, of in particular myelomonocytic differentiation. In contrast, the receptor for SF, i.e., Kit, is highly expressed on very immature CD34-bright/HLA-DR-dull cells, which include stem cells. Kit levels decline during myelomonocytic and B-lymphoid differentiation whereas they increase to maximal levels during early stages of erythropoiesis. The heterogeneity in receptor expression, together with other immunophenotypic characteristics, allows for the identification of distinct progenitor cell subsets and differentiation stages within the CD34+ cell compartment. By selecting appropriate phenotypic criteria it will be possible to further dissect the stem cell compartment and eventually establish the, possibly heterogeneous, HGF receptor phenotype of pluripotent stem cells.

Purification of repopulating hemopoietic cells based on binding of biotinylated Kit ligand.

To characterize Kit expressing mouse bone marrow (BM) cells, and to determine their contribution to short- and long-term repopulation of the hemopoietic system of irradiated recipients, we have purified Kit+ BM cells by flow cytometry. A high level of Kit expression was detectable on 1-2% of BM cells after staining with biologically active biotinylated Kit ligand (KL) or with anti-Kit antibodies (ACK-2). Compared to unfractionated BM, the Kit+ fractions were enriched for immature hemopoietic cells, as shown by morphological differentiation, in vitro culture, and spleen colony formation. Enrichment of colony-forming cells was higher in biotin-KL+ than ACK-2+ fractions. Colony-forming cells were not found in the Kit- subsets. To study the hemopoietic repopulation capacity of the Kit+ and Kit- cells, serial dilutions of the sorted fractions were transplanted into irradiated mice, and peripheral blood of these recipients was monitored regularly for the presence of donor-derived cells during a 1 year period. Nucleated blood cell repopulation by male donor cells in female recipients was assessed using a Y-chromosome specific DNA probe; erythroid repopulation by normal donor cells in W/Wv recipients was examined flow cytometrically by measuring the forward light scatter of donor- and host-type erythrocytes. A 25- to 100-fold enrichment of long-term repopulating ability in the sorted Kit+ fractions showed that Kit+ cells are capable of reconstitution of circulating erythrocytes and nucleated blood cells after BM transplantation. Transient repopulation of the red blood cell lineage was observed after transplantation of Kit- cells. Detection of donor-derived nucleated cells 1 year after transplantation showed that Kit+ cells contributed to donor-type repopulation of bone marrow, spleen and thymus. Our data demonstrate that isolation of BM cells on the basis of Kit expression is a useful addition to the methods that are commonly applied in stem cell enrichment protocols.

Separation of myeloid and erythroid progenitors based on expression of CD34 and c-kit.

In this report, a novel approach is described to physically separate erythroid progenitors from monocyte and granulocyte progenitors, based on the expression of CD34 and Kit. Using biotin-labeled human Kit ligand (KL) and flow cytometry, Kit was detectable on 2% to 3% of the nucleated cells in rhesus monkey bone marrow. Combination of biotin-KL with CD34 monoclonal antibodies (MoAb) showed that Kit was expressed on subsets of CD34low and CD34pos cells. Our data clearly demonstrate that CD34pos cells are more heterogeneous with respect to Kit expression than observed in studies using Kit MoAb. A small cluster, approximately 7% of the CD34pos cells, expressed CD34 at submaximal levels and stained brightly with biotinylated KL. This CD34pos/kithi fraction contained predominantly erythroid progenitors (burst-forming units-erythroid; BFU-E). The majority of the granulocytic and monocytic progenitors (colony-forming units-granulocyte/macrophage; CFU-GM) were CD34pos/kitmed. Some BFU-E were also detected in the CD34pos/kitmed and CD34low/kitpos fractions at low frequency. In the latter subset, most erythroid colony-forming units (CFU-E) were recovered. Using three-color flow cytometry, we analyzed expression of Kit in relation to that of CD34 and the class II major histocompatibility antigen, RhLA-DR. The most immature bone marrow cells that can be identified in vitro, ie, CD34pos/RhLA-DRlow cells, were kitmed. The CD34pos/kithi and CD34pos/kitneg subsets predominantly contained the more mature RhLA-DRbright cells. Our results demonstrate that erythroid precursors express c-kit at much higher levels than monomyeloid precursors and pluripotent progenitors. The difference in expression levels of CD34 and c-kit can be exploited to isolate BFU-E populations that are virtually devoid of nonerythroid cells.

Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression. Comparison of different methods.

The main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that binding of the biotinylated ligand to the receptor does not inhibit the subsequent interaction of biotin with fluorescently tagged avidin or streptavidin. Using interleukin-2 (IL-2), we compared the usefulness of various biotinylation reagents, NHS-biotin, S-NHS-biotin, S-NHS-LC-biotin, DBB and photobiotin, and developed optimal biotinylation conditions for the preparation of biologically active biotin-labeled IL-2 and the detection of IL-2 receptor expressing cells by flow cytometry. As determined by spot blot analysis, biotinylation of IL-2 was most efficient at the highest biotin-to-protein (B:P) ratio used. At a B:P ratio of 100, most of the biological activity of IL-2 was retained when S-NHS-LC-biotin was used. In contrast, most of the biological activity of IL-2 samples that were labeled with NHS-biotin or photobiotin was lost under these conditions. Biotin-labeled IL-2 preparations were tested in order to detect IL-2 receptors on IL-2 dependent CTLL-2 cells by flow cytometry after sequential staining with the biotinylated IL-2 and fluorescence tagged streptavidin. A high B:P ratio generally resulted in a high specific fluorescence intensity of the cells, particularly when S-NHS-LC-biotin was used as the biotinylation reagent. Biotin-IL-2 could also be used to detect IL-2 receptors expressed by lymphocytes in peripheral blood and bone marrow. Comparison of staining of lymphocytes with biotinylated IL-2 and an antibody against the IL-2 receptor alpha chain demonstrated that only a subset of the cells that showed a strong fluorescence signal after staining with biotinylated IL-2 expressed high numbers of the IL-2 receptor alpha chain. This is in agreement with the expression of functional IL-2 receptors on resting T cells and NK cells which do not express the alpha chain. After stimulation with PHA, virtually all lymphocytes expressed the alpha chain, whereas only part of these cells showed a strong fluorescence signal after staining with biotin-IL-2, while the rest of the cells had very low numbers of IL-2 binding sites. Our results demonstrate that, in addition to staining individual receptor subunits with antibodies, staining with biotinylated IL-2 is a useful indicator of functional IL-2 receptor expression.

Differential expression of receptors for interleukin-3 on subsets of CD34-expressing hematopoietic cells of rhesus monkeys.

The target cell specificity of interleukin-3 (IL-3) was examined by flow cytometric analysis of IL-3 receptor (IL-3R) expression on rhesus monkey bone marrow (BM) cells using biotinylated IL-3. Only 2% to 5% of unfractionated cells stained specifically with the biotinylated IL-3 and most of these cells were present within the CD34+ subset. IL-3Rs were detected on small CD34dull/RhLA-DRbright/CD10+/CD27+/CD2-/++ +CD20- cells, which probably represent B-cell precursors. IL-3R+ CD34- BM cells, which were detected at low frequencies, consisted of small CD20dull/surface-IgM+/RhLA-DR+ cells. These cells represented immature B lymphocytes, whereas CD20bright mature B cells were IL-3R-. The highest IL-3R levels were detected on CD34dull/RhLA-DRbright blast-like cells. These cells differentiated into monocytes, neutrophils, and basophils after IL-3 and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation in vitro. The CD34bright/IL-3R- subset contained all clonogenic erythroid and myeloid progenitors (burst-forming unit-erythroid and colony-forming unit-culture), whereas CD34bright/IL-3Rdull cells differentiated into monocytes, neutrophils, and erythroid cells after shorter culture periods. This finding showed that IL-3R expression increases during monocyte and granulocyte differentiation. Results of three-color experiments indicated that IL-3Rs are expressed on CD34bright/RhLA-DRbright cells as well as on CD34bright/RhLA-DRdull cells, with the latter population expression approximately twofold to threefold lower IL-3R levels. A large fraction (> 30%) of single-cell/well-sorted CD34bright/RhLA-DRdull cells formed multilineage colonies after 2 to 4 weeks of stimulation with IL-3, GM-CSF, Kit ligand, and IL-6. Individual colonies contained cells that still expressed CD34 as well as differentiated monocytes, granulocytes, and erythroid cells. These results confirmed that the CD34bright/RhLA-DRdull subset was enriched for immature, multipotent progenitor cells, whereas the CD34bright/RhLA-DRbright population mainly contained lineage-committed precursors. The results are consistent with the concept that IL-3Rs are induced at very early stages of hematopoiesis, as identified by high expression of CD34 and low expression of RhLA-DR. IL-3R expression continues to be low during differentiation into lineage-committed progenitors; gradually increases on differentiating progenitor cells for B cells, granulocytes, monocytes, and, possibly also, erythrocytes; but finally declines to undetectable levels during terminal differentiation into mature cells of all lineages in peripheral blood, with the exception of basophils.(ABSTRACT TRUNCATED AT 400 WORDS)

The expression of cytokine receptors by purified hemopoietic stem cells.

Sorted fractions from mouse bone marrow containing highly purified hemopoietic stem and progenitor cells were studied for the expression of growth factor receptors. With the use of rhodamine 123 WGA+, 15-1.1-, low density cells were separated into quiescent pluripotent stem cells and committed progenitor cells. RNA was extracted and cDNA was prepared by reverse transcription. Using primers specific for growth factor receptors, the cDNA of each sorted fraction was amplified by polymerase chain reaction (PCR). The quiescent rhodamine 123 dull stem cell fraction was found to express the interleukin 3 (IL-3) receptor beta unit and c-kit, but not the granulocyte-macrophage colony stimulating factor (GM-CSF) receptor beta unit nor flk-2. The rhodamine 123 bright fraction with activated stem cells and mostly committed progenitor cells similarly expressed the IL-3-R beta, and c-kit. However, this fraction also expressed flk-2 and GM-CSF-R beta. Since the expression of c-kit in the stem cell fraction does not correspond with the poor response to the kit-ligand stem cell factor (SCF) by these cells, we further analyzed the fractions with respect to their binding of biotinylated SCF. The SCF-binding cells were found to be all rhodamine 123 bright. This indicates that the expression of c-kit is not sufficient to yield a functional receptor for SCF; c-kit probably needs a partner molecule to form a functional high-affinity binding site for SCF. Similar to the beta unit of the GM-CSF receptor, this partner is then not expressed in the stem cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)