Action of interleukin-3, G-CSF, and GM-CSF on highly enriched human hematopoietic progenitor cells: synergistic interaction of GM-CSF plus G-CSF

Purified preparations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and interleukin 3 (IL-3 or multi-CSF) alone and in combination, have been compared for their stimulatory effects on human granulocyte-macrophage colony forming cells (GM-CFC). In cultures of unseparated normal human bone marrow, the combinations of G-CSF plus IL-3 and GM-CSF plus IL-3 stimulated additive numbers of GM colonies, while GM-CSF plus G-CSF stimulated greater than additive numbers of GM colonies, compared with the sum of the colony formation obtained with each factor alone. Cultures of unseparated bone marrow, harvested from patients four to six days after administration of 5-fluorouracil (5-FU), resulted in additive GM colony formation with GM-CSF plus G-CSF, GM-CSF plus IL-3, and G-CSF plus IL-3. In order to address the possibility of secondary factor involvement in the synergistic interaction of GM-CSF and G-CSF, CD33+/CD34+ colony forming cells were separated from normal and post FU marrow by two color fluorescence activated cell sorting. In cultures of CD33+/CD34+ cells the combination of GM-CSF plus G-CSF stimulated a synergistic increase in GM colonies while GM-CSF plus IL-3 stimulated additive numbers of colonies. These results suggest that GM-CSF, G-CSF, and IL-3 stimulate distinct populations of GM-CFC. Furthermore GM-CSF and G-CSF interact synergistically and this action is a direct effect on progenitor cells not stimulated by GM-CSF or G-CSF alone.     

Characterization of a factor-dependent acute leukemia cell line with translocation (3;3)(q21;q26).

A strictly factor-dependent cell line (UCSD/AML1) was established from a patient with the syndrome of multilineage acute leukemia with high platelets. The patient's cells and the cell line karyotype were 45,XX,-7,t(3;3)(q21;q26), typical of the syndrome of acute leukemia with high platelets. The cell line expresses CD34, CD7, TdT, and myeloid (CD13, CD14, CD33) and megakaryocyte/platelet (CD36, CD41, CD42b, CDw49b) antigens. In short-term culture, UCSD/AML1 cells proliferate in response to interleukin-3 (IL-3), IL-4, IL-6, macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage CSF (GM-CSF), but not IL-1, IL-2, IL-5, or G-CSF. In long-term culture, proliferation can be sustained by GM-CSF, IL-6, or M-CSF. When maintained in GM-CSF, a small percentage of cells form multinucleated megakaryocyte-like giant cells. Culture with GM-CSF combined with IL-6, but not with IL-6 alone, increased giant cell formation fourfold to sevenfold. IL-6 alone or in combination with GM-CSF increased expression of platelet-related antigens. In contrast, culture with phorbol ester induced formation of macrophage-like cells. UCSD/AML1 is the first human acute nonlymphocytic leukemia cell line established from a patient with an acute leukemia syndrome associated with a specific chromosome abnormality.     

Initiation in DNA synthesis by colony-stimulating factors in subsets of human CD34+ marrow cells.

Initiation of DNA synthesis by recombinant colony-stimulating factors (CSFs) was assessed in normal human marrow blast cells isolated by expression of CD34 antigen (tritiated thymidine incorporation). Continuous exposure to CSF was required. A mild increase in DNA synthesis was initiated by granulocyte CSF (G-CSF; greater than or equal to 1 ng/ml), to approximately 1.5 times control levels. A greater increase was initiated by granulocyte-macrophage CSF (GM-CSF), with a threshold of approximately 0.1 ng/ml and a plateau increment 2.5 times control levels. CD34+ cells were stimulated by interleukin 3 (IL-3) over a wide concentration range: two times control at 0.1/ml, three times control at 1 ng/ml, and four times control at 10 ng/ml. Overlap between responding populations was analyzed. G-CSF plus GM-CSF induced DNA synthesis greater than GM-CSF alone and supported the growth of much larger granulocyte-monocyte colonies. At saturating IL-3 concentrations, neither G-CSF nor GM-CSF induced additional DNA synthesis; at lower concentrations of IL-3, however, GM-CSF recruited additional cells into DNA synthesis. Using CD10 and CD19 antibodies to separate B-lineage cells, the CD34+ cells responding to CSF were observed to be in the non-B-lineage subset. Therefore 1) the response of CD34+ cell subsets CSFs is IL-3 greater than GM-CSF greater than G-CSF, and the IL-3-responsive population is heterogeneous for dose requirement; 2) a CD34+ subpopulation responding to concurrent G-CSF and GM-CSF includes increased proliferative potential cells; 3) IL-3-responsive cells include GM-CSF- and G-CSF-responsive cells, but cells responding to lower IL-3 concentration do not respond to GM-CSF; and 4) B-cell precursors do not respond to GM-CSF or IL-3 in this assay.     

Growth regulation of human acute myeloid leukemia: effects of five recombinant hematopoietic factors in a serum-free culture system

The response of human acute myeloid leukemia (AML) cells to the distinct hematopoietic growth factors (HGFs), ie, recombinant interleukin-3 (IL-3), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), and erythropoietin (Epo) was investigated under well-defined serum-free conditions. Proliferative responses to these factors, when added separately as well as in combinations, were analyzed in 25 cases of human AML using 3H-thymidine incorporation and colony assays. The 3H-thymidine uptake data revealed that IL-3, GM-CSF, G-CSF, and M-CSF were stimulators of AML proliferation in 19, 15, 13, and 4 cases, respectively. Epo only stimulated DNA synthesis in the cells of the single erythroleukemia case. GM-CSF stimulation was seen only in IL-3 reactive cases and GM-CSF, when combined with IL-3, could not further elevate the DNA synthesis evoked by IL-3 alone. On the other hand, in six cases, G-CSF enhanced the IL-3- or GM-CSF-stimulated thymidine uptake. These results suggest that subpopulations of AML cells that are activated by distinct CSFs (eg, IL-3/GM-CSF-responsive cells and G-CSF-responsive cells) coexist. The 3H-thymidine incorporation assay was more sensitive for measuring CSF responses than methylcellulose colony cultures, since activation of DNA synthesis was more frequently seen than induction of colony formation. DNA synthesis experiments revealed eight different CSF response patterns among these 25 cases. CSF phenotyping may be a useful addition to the morphologic classification of AML, since these patterns directly reflect the ability of the proliferating subsets of AML cells to respond to the CSFs.     

Effects of recombinant human G-CSF, GM-CSF, IL-3, and IL-1 alpha on the growth of purified human peripheral blood progenitors.

The effects of recombinant products of granulocyte colony-stimulating factors (G-CSF), granulocyte/macrophage CSF (GM-CSF), human interleukin-3 (IL-3), and interleukin-1 (IL-1) were studied using purified target cell populations from patients undergoing peripheral blood stem cell transplantation after myeloablative therapy. Cells were subjected to combined purification procedures including negative selection with a panel of monoclonal antibodies (CD2, 3, 5, 10, and 20). The purified cells were enriched for HLA-DR+ (51% to 71%) and My-10+ (CD34; 37% to 54%) and had a plating efficiency of up to 20%. In the liquid-suspension limiting dilution clonal assay (LDA), purified progenitors responded directly to IL-3 by proliferation with single-hit kinetics. The ability of GM-CSF to support progenitor growth was inferior to that of IL-3, and the cells were virtually unresponsive when cultured with G-CSF, supporting the notion that these blood-derived progenitors belong to a primitive population of hematopoietic progenitor cells. The results obtained in simultaneous methycellulose cultures (MC) showed the same trend and provided additional information on the ability of GM-CSF and IL-3 to support erythroid progenitor growth. The combination of IL-3 plus G-CSF, but not IL-3 plus GM-CSF, resulted in a synergistic increase in colony number. IL-1 alpha increased both the size and number of colonies when added to IL-3 or G-CSF. Study of this enriched progenitor cell population in MC and LDA represents an excellent model for the investigation of myeloid and erythroid differentiation and for evaluating the influence of various cytokines on human hematopoiesis.     

Cytokine-induced selective expansion and maturation of erythroid versus myeloid progenitors from purified cord blood precursor cells

To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells, a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells, purified from umbilical cord blood by flow cytometry and cell sorting, were selected for this study because of their high content of clonogenic cells (34%), particularly multipotent progenitors (CFU-MIX, 12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF, IL-6, IL-3, and erythropoietin (Epo); (3) MGF, IL-6, granulocyte-macrophage colony- stimulating factor (GM-CSF)/IL-3 fusion protein (FP), macrophage colony- stimulating factor (M-CSF), and granulocyte-CSF (G-CSF); and (4) MGF, IL-6, FP, M-CSF, G-CSF, and Epo. Maximum numbers of erythroid progenitors (BFU-E, up to 55-fold increase) and mature erythroid cells were observed in the presence of MGF, IL-6, IL-3, and Epo, whereas maximum levels of myeloid progenitors (CFU-C, up to 70-fold increase) and mature myeloid cells were found in cultures supplemented with MGF, IL-6, FP, M-CSF, and G-CSF. When MGF, IL-6, FP, M-CSF, G-CSF, and Epo were present, maximum levels of both erythroid and myeloid progenitors and their progeny were observed. These results indicate that specific cytokine combinations can act directly on primitive hematopoietic cells resulting in significant expansion of progenitor cell numbers and influencing their overall patterns of proliferation and differentiation. Furthermore, the observations presented in this study suggest that the cytokine combinations used were unable to bias lineage commitment of multipotent progenitors, but rather had a permissive effect on the development of lineage-restricted clonogenic cells.     

Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor.

We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.     

Regulation of differentiation of murine progenitor cells derived from blast cell colonies under serum-deprived conditions

We have examined the effect of interleukin 3 (IL-3), granulocyte-macrophage (GM)-, granulocyte (G)-, and macrophage (M)-colony-stimulating factors (CSFs) on the induction of GM colonies from highly enriched murine hematopoietic progenitor cells under serum-deprived conditions. Each growth factor was tested alone or in combination with suboptimal concentrations of the others. The effect of each CSF on GM colony growth in fetal bovine serum (FBS)-supplemented cultures of unfractionated marrow cells is reported for comparison. GM-CSF induced GM colony growth in serum-deprived cultures of purified progenitor cells to the same extent as in FBS-supplemented cultures of unfractionated marrow cells. In contrast, IL-3 was only one-tenth as active in promoting the growth of enriched progenitor cells under serum-deprived conditions when compared with its effect on colony growth from unfractionated marrow. M-CSF and G-CSF were almost completely ineffective in both cases. G-CSF induction of GM colony growth from purified progenitor cells was restored by addition of suboptimal concentrations of IL-3 or GM-CSF, suggesting that either IL-3 or GM-CSF is required to observe the effect of G-CSF. Addition of G-CSF to GM-CSF-stimulated cultures did not increase the maximal number of colonies detected, indicating that these two growth factors may act on the same subset of progenitor cells. Addition of GM-CSF or IL-3 to IL-3- or GM-CSF-stimulated cultures, respectively, increased by 40% the maximal number of colonies detected, suggesting that these two factors act on at least partially separate subsets of GM progenitors. These data parallel the recent observations on the control of human GM colony formation under FBS-deprived conditions and support a model for the control of myeloid differentiation that requires the interplay of different growth factors.     

Sequential loss of CD34 and class II MHC antigens on purified cord blood hematopoietic progenitors cultured with IL-3: characterization of CD34-, HLA-DR+ cells

The expression of class II MHC and CD34 antigens on human cord blood hematopoietic progenitor cells (HPC) was investigated upon culturing in the presence of interleukin-3 (IL-3). HPC isolated by "panning" according to their expression of CD34 coexpressed HLA-DR and HLA-DP, and the majority of the CD34+ HPC also expressed HLA-DQ. In the presence of IL-3, the expression of CD34 and class II MHC antigens was found to be gradually lost in culture. Loss of CD34 expression preceded loss of HLA-DR expression. After eight days of culture, CD34-, HLA-DR+ blast cells were obtained that strongly proliferated in response to IL-3, GM-CSF, G-CSF, and M-CSF, and that had the capacity to generate macrophage and granulocyte colonies. After ten days of culture in IL-3, a population of CD34- cells that expressed low levels of HLA-DR (HLA-DRlo) was obtained by FACS-sorting. These CD34-, HLA-DRlo cells lacked colony-forming activity while the population expressing high levels of HLA-DR (HLA-DRhi) contained great numbers of colony-forming cells, and proliferated stronger in response to CSFs than the HLA-DRlo fraction. Finally CD34-, HLA-DR- cells that appeared later in the cultures (14 to 16 days) represented more differentiated cells with only marginal proliferative and no clonogenic capacity. These data indicate that whereas CD34 expression is associated with the multilineage potential of the HPC, HLA-DR expression correlates with overall proliferative capacity of hematopoietic cells during culture in IL-3.     

Interleukin-1, tumor necrosis factor, and the production of colony- stimulating factors by cultured mesenchymal cells

Although the genes for four hematopoietic colony-stimulating factors (CSFs) have been cloned, neither the mechanism of the regulation of their production nor their cellular origins have been established with certainty. Monocytes are known to produce colony-stimulating and burst- promoting activities, as well as several monokines such as interleukin- 1 (IL-1) and tumor necrosis factor (TNF). These monokines indirectly stimulate other mesenchymal cells to produce certain colony-stimulating factors such as granulocyte-macrophage CSF (GM-CSF). To determine whether monocytes produce other CSFs and if so, to compare the mechanism of regulation of production with that of endothelial cells and fibroblasts, we investigated the synthesis of CSFs by monocytes, human umbilical vein endothelial cells, and fibroblasts. We used total cellular RNA blot analysis to determine interleukin-3 (IL-3), GM-CSF, granulocyte CSF (G-CSF), and monocyte CSF (M-CSF) messenger RNA (mRNA) content and immunoprecipitation or bioassay to confirm the presence of the specific secreted proteins. The results indicate that M-CSF mRNA and protein are produced constitutively by all three cell types and their level of expression does not increase after induction. In contrast, GM-CSF and G-CSF mRNAs are barely detectable in uninduced monocytes and show an increase in expression after lipopolysaccharide treatment. Retrovirus-immortalized endothelial cells, unlike primary endothelial cells or both primary and immortalized fibroblasts, produce IL-1 constitutively; this correlates with their constitutive production of GM-CSF and G-CSF. IL-3 mRNA was not detectable in any of these cells either before or after induction. The results indicate that these mesenchymal cells can produce three CSFs: GM-CSF, G-CSF, and M-CSF; furthermore, the data suggest that the mechanism of regulation of M-CSF production is different from that of GM-CSF and G-CSF, and that the latter two inducible CSFs are regulated by different factors in monocytes compared with the other mesenchymal cells.     

Combined and sequential effects of human IL-3 and GM-CSF on the proliferation of CD34+ hematopoietic cells from cord blood

The proliferative effects of recombinant human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were investigated in semi-solid and liquid cultures of purified CD34+ hematopoietic cells obtained from umbilical cord blood. No important differences in overall cloning efficiencies in response to IL-3 or GM- CSF were observed in semi-solid medium in the presence of erythropoietin (Ep). However, GM-CSF was less effective for the development of erythroid bursts (BFU-E), and only IL-3 was observed to induce significant numbers of mixed-erythroid colonies (E-MIX). Both IL- 3 and GM-CSF also induced proliferation of CD34+ in liquid cultures. Proliferative responses to IL-3 were found to be more rapid and stronger than to GM-CSF, although the number of initial responsive cells as judged by autoradiography were comparable. Enhanced proliferation of CD34+ cells both in semi-solid and liquid cultures was obtained in the presence of combinations of IL-3 and GM-CSF. The responses observed were less than additive, with the exception of the development of eosinophil colonies and clusters, where IL-3 and GM-CSF were found to act synergistically. In secondary cultures, proliferative responses to GM-CSF were strongly enhanced by preculture of CD34+ cells in IL-3 for four to 11 days, and to a lesser extent by preculture in GM- CSF. Finally, responses to IL-3 were not affected by preculture of CD34+ cells in the presence of GM-CSF. Our results indicate that there is a wide overlap of cells capable of proliferating either in response to IL-3 or to GM-CSF within the cord blood CD34+ compartment. However, differences in primary proliferation kinetics and increased responsiveness to GM-CSF following preculture suggest the importance of a sequential action of IL-3 and GM-CSF in the expansion of CD34+ cells.     

Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro.

An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.     

The FLT3 ligand is a direct and potent stimulator of the growth of primitive and committed human CD34+ bone marrow progenitor cells in vitro

The present studies investigated the effects of the recently cloned flt3 ligand (FL) on the in vitro growth and differentiation of primitive and committed subsets of human CD34+ bone marrow (BM) progenitor cells. FL alone was a weak growth stimulator of CD34+ BM cells, but synergistically and directly enhanced colony formation in combination with interleukin (IL) 3, granulocyte colony-stimulating factor (G-CSF), CSF-1, granulocyte macrophage (GM) CSF stem cell factor (SCF), and IL-6. FL and SCF were equally effective in stimulating colony formation in combination with IL-3. However, the tri-factor combination of FL + IL-3 + SCF stimulated 2.3-fold and 2.5-fold more colonies than FL + IL-3 and SCF + IL-3, respectively. These additional recruited progenitors appeared to be predominantly located in a primitive (CD71-) subset of the CD34+ progenitors, as 4.5-fold more colonies were formed by CD34+CD71- cells in response to FL + IL-3 + SCF than to FL + IL-3 or SCF + IL-3. Similar findings were observed in serum-containing and serum-deprived cultures. Whereas FL did not enhance burst-forming unit-erythroid (BFU-E) colony formation of CD34+ BM cells in the presence of serum, a low number of BFU-E colonies were formed in response to FL plus erythropoietin (Epo) under serum-deprived conditions. In addition, FL both in serum-containing and serum-deprived cultures stimulated colony formation of more committed myeloid progenitors in CD34+CD71+ BM cells. Thus, FL potently stimulates the growth of primitive and more committed human BM progenitor cells.     

Expansion of granulocyte colony-stimulating factor/chemotherapy-mobilized CD34+ hematopoietic progenitors: role of granulocyte-macrophage colony-stimulating factor/erythropoietin hybrid protein (MEN11303) and interleukin-15

Ex vivo stroma-free static liquid cultures of granulocyte colony-stimulating factor (G-CSF)/chemotherapy-mobilized CD34+ cells were established from patients with epithelial solid tumors. Different culture conditions were generated by adding G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt3 ligand (Flt3), megakaryocyte growth and development factor (Peg-rHuMGDF), GM-CSF/erythropoietin (EPO) hybrid protein (MEN11303), and interleukin-15 (IL-15) to the basic stem cell factor (SCF) + interleukin-3 (IL-3) + EPO combination. This study showed that, among the nine different combinations tested in our 5% autologous plasma-containing cultures, only those containing IL-3/SCF/Flt3/MEN11303 and IL-3/SCF/Flt3/MEN11303/IL-15 significantly expanded colony-forming unit granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), long-term culture-initiating cells (LTC-IC), CD34+, and CD34+/CD38- cells after 14 days of culture. Particularly, the addition of IL-15 to IL-3/SCF/Flt3/MEN11303 combination produced a significant increase of LTC-IC, with an average 26-fold amplification as compared to input cells, without any detrimental effect on CFU-GM and BFU-E expansion. This combination also produced a statistically significant 3.6-fold expansion of primitive CD34+/CD38- cells. Moreover, this study confirms the previously described erythropoietic effect of MEN11303, which, in our experience, was the only factor capable of expanding BFU-E. Compared to equimolar concentrations of GM-CSF and EPO, MEN11303 hybrid protein showed a significantly higher capacity of expanding CFU-GM, BFU-E, LTC-IC, CD34+, and CD34+/CD38- cells when these cytokines were tested in combination with IL-3/SCF/Flt3. These cultures indicated that Peg-rHuMGDF addition to IL-3/SCF/EPO/Flt3 does not affect CFU-GM and BFU-E expansion but, unlike G-CSF or GM-CSF, it does not decrease the ability of Flt3 to expand primitive LTC-IC. These studies indicate that, starting from G-CSF/chemotherapy-mobilized CD34+ cells, concomitant expansion of primitive LTC-IC, CFU-GM, BFU-E, CD34+, and CD34+/CD38- cells is feasible in simple stroma-free static liquid cultures, provided IL-3/SCF/Flt3/MEN11303/IL-15 combination is used as expanding cocktail in the presence of 5% autologous plasma.     

Regulation of colony forming cell generation by flt-3 ligand

The recently cloned ligand for the flt-3/flk-2 receptor was examined for its effect on colony formation by subpopulations of CD34⁺ cells including the least mature CD34⁺lin⁻CD38⁻ small-medium lymphocyte-sized cell population. Flt-3 ligand (flt-3l) had little or no effect when added alone to cells. Isolated CD34⁺lin⁺ cells formed increased numbers of colony-forming cells (CFC) when flt-3l was added together with IL-3, IL-6, G-CSF, GM-CSF or c-kit ligand (KL), or with the combination of IL-3 and KL. Significant increases in CFC formation from CD34⁺lin⁻ cells were consistently seen when flt-3l was added to the IL-3 and KL combination, with variable effects observed when it was added to individual growth factors. Studies of the generation of CFC from CD34⁺lin⁻ cells in liquid cultures showed that cultures containing IL-3 and KL continued to produce CFC after 3 weeks of culture, whereas cultures with IL-3, KL and flt-3l produced few CFC past 2 weeks of culture. Flt-3l alone or the combination of IL-3 and KL did not stimulate significant growth of CD34⁺lin⁻CD38⁻ small-medium lymphocyte-sized cells, although these cells reproducibly generated CFC when grown in the combination of IL-1β, IL-3, IL-6, G-CSF, GM-CSF and KL. Addition of flt-3l to either IL-3 and KL or to a combination of growth factors induced increased CFC in three of four experiments. These data therefore demonstrate a role for flt-3l in the induction of myelopoiesis by haemopoietic precursors, including the least mature subpopulation population of CD34⁺ cells.     

Malignancy: Insulin-like Growth Factor-1 (IGF-1) is a Costimulator of the Expansion of Lineage Committed Cells Derived from Peripheral Blood Mobilized CD34+ Cells in Multiple Myeloma Patients

The effect of insulin-like growth factor-1 (IGF-1) on highly enriched human apheresis CD34(+) progenitor cells was investigated in vitro. The progenitor cells were mobilized by treatment with cyclophosphamide + granulocyte - colony stimulating factor (G-CSF) in patients with multiple myeloma. CD34(+) cells were cultured for 7 days in serumfree medium containing stem cell factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), and this is referred to as cytokine-dependent proliferation. After 7 days of cytokine-dependent proliferation the total number of viable cells increased 1.6-8.2 times, and subsets of cells expressing the granulocyte marker CD15, the myelomonocytic marker CD64 and the erythrocyte phenotype CD71(high) /CD64(-) were detected among the in vitro cultured cells. Addition of G-CSF together with SCF + IL-3 + GM-CSF increased the number of CD15(+) and CD64(+) cells, but without altering the number of erythroid cells. IGF-1 caused a dose-dependent increase in the number of CD15(+), CD64(+) and CD71(high) /CD64(-) cells, and this increase was detected when cells were cultured in both SCF + IL-3 + GM-CSF alone and G-CSF + SCF + IL-3 + GM-CSF. A minor subset of CD34(+) cells could still be detected among in vitro cultured cells and the number of CD34(+) cells was not altered by adding G-CSF and/or IGF-1. Morphologically recognizable mature granulocytes or erythroid cells could not be detected for any of the combinations investigated. We conclude that IGF-1 can enhance the in vitro proliferation of committed progenitor cells derived from apheresis CD34(+) cells.     

Megakaryopoiesis in vitro in myelodysplastic syndromes and acute myeloid leukaemia: effect of pegylated recombinant human megakaryocyte growth and development factor in combination with other growth factors

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) can stimulate megakaryopoiesis in vitro in some myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients. We assessed PEG-rHuMGDF combined with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3), IL6, stem cell factor (SCF) or erythropoietin in 40 MDS, 33 AML and 16 normal bone marrow samples. CD61-positive cells in suspension cultures increased with PEG-rHuMGDF alone in 20/25 RA + RAS, 11/14 RAEB + RAEBt and 29/33 AML cases. Further increases when IL-3 and/or SCF were added to PEG-rHuMGDF occurred in 14/20 RA + RAS, 8/13 RAEB + RAEBt and 18/26 AML cases. CFU-Mk growth was poor overall, but could be enhanced by PEG-rHuMGDF combinations in some patients. Stimulation of megakaryopoiesis by PEG-rHuMGDF can be augmented by IL-3 and SCF in many MDS and AML patients.     

Hematopoietic recovery following high-dose combined alkylating-agent chemotherapy and autologous bone marrow support in patients in phase-I clinical trials of colony-stimulating factors: G-CSF,GM-CSF,IL-1, IL-2, M-CSF

Summary Hematopoietic recovery in 115 patients with metastatic breast cancer or metastatic melanoma, enrolled in phase-I studies of recombinant growth factors while undergoing treatment with high-dose chemotherapy with autologous bone marrow support, was examined with assays of bone marrow progenitor cells and peripheral blood progenitor cells, and by evaluation of peripheral blood counts. Groups of patients receiving hematopoietic cytokine support [with interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or monocyte CSF (M-CSF)] post marrow infusion were compared with contemporaneous control patients not receiving growth factor support. Patients receiving GM-CSF demonstrated statistically significant increases in the growth of granulocyte/macrophage colony-forming units (CFU-GM) in the bone marrow and peripheral blood compared with control patients. The effect of GM-CSF was dose dependent in the early period post marrow infusion (day +6) with bone marrow CFU-GM colonies at doses 8–16 g/kg/ day 34 times those measured in controls. Significant increases in bone marrow multipotential progenitor cells (CFU-GEMM) were seen in patients receiving GMCSF day + 21 post marrow infusion. Patients receiving IL-1 demonstrated significant increases in bone marrow CFU-GM at day +21, maximal at dosages of 24–32 ng/kg/day. There were no significant increases in burst forming unit-erythroid (BFU-E) among any study group. Patients receiving G-CSF had significantly increased absolute neutrophil counts (ANC) and total white blood cell counts (WBC) by day +11 post transplant compared with control patients. Patients receiving GM-CSF demonstrated significantly increased WBC (greater than 2000/mm³) at day +11 and ANC greater than 500/mm³ at day +16. Optimal dose of GCSF and GM-CSF to stimulate neutrophil recovery post transplant was 4–8 g/kg/day and 8–16 g/kg/day, respectively. Platelet recovery did not differ among the six study groups. These data demonstrate accelerated myeloid recovery after high-dose chemotherapy and autologous bone marrow support in patients receiving either G-CSF or GM-CSF. Moreover, GM-CSF and IL-1 stimulate myelopoiesis at the level of bone marrow CFU-GM, while G-CSF causes earlier neutrophil recovery peripherally.This work has been supported in part by The National Heart, Lung, and Blood Institute, grant P01CA47741. Joanne Kurtzberg, MD is a scholar of the Leukemia Society of America     

A comparative study of the phenotype and proliferative capacity of peripheral blood (PB) CD34+ cells mobilized by four different protocols and those of steady-phase PB and bone marrow CD34+ cells

Peripheral blood (PB) CD34+ cells from four commonly used mobilization protocols were studied to compare their phenotype and proliferative capacity with steady-state PB or bone marrow (BM) CD34+ cells. Mobilized PB CD34+ cells were collected during hematopoietic recovery after myelosuppressive chemotherapy with or without granulocyte- macrophage colony-stimulating factor (GM-CSF) or granulocyte colony- stimulating factor (G-CSF) or during G-CSF administration alone. The expression of activation and lineage-associated markers and c-kit gene product were studied by flow cytometry. Proliferative capacity was measured by generation of nascent myeloid progenitor cells (granulocyte- macrophage colony-stimulating factor; CFU-GM) and nucleated cells in a stroma-free liquid culture stimulated by a combination of six hematopoietic growth factors (interleukin-1 (IL-1), IL-3, IL-6, GM-CSF, G-CSF, and stem cell factor). G-CSF-mobilized CD34+ cells have the highest percentage of CD38- cells (P < .0081), but otherwise, CD34+ cells from different mobilization protocols were similar to one another in their phenotype and proliferative capacity. The spectrum of primitive and mature myeloid progenitors in mobilized PB CD34+ cells was similar to their steady-state counterparts, but the percentages of CD34+ cells expressing CD10 or CD19 were lower (P < .0028). Although steady-state PB and chemotherapy-mobilized CD34+ cells generated fewer CFU-GM at day 21 than G-CSF-mobilized and steady-state BM CD34+ cells (P < .0449), the generation of nucleated cells and CFU-GM were otherwise comparable. The presence of increased or comparable numbers of hematopoietic progenitors within PB collections with equivalent proliferative capacity to BM CD34+ cells is not unexpected given the rapid and complete hematopoietic reconstitution observed with mobilized PB. However, all four types of mobilized PB CD34+ cells are different from steady-state BM CD34+ cells in that they express less c-kit (P < .0002) and CD71 (P < .04) and retain less rhodamine 123 (P < .0001). These observations are novel and suggest that different mobilization protocols may act via similar pathways involving the down-regulation of c-kit and may be independent of cell-cycle status.