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.     

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) on megakaryopoiesis in patients with aplastic anaemia

Recombinant human megakaryocyte growth and development factor (rHuMGDF), a truncated form of the Mpl ligand, stimulates megakaryopoiesis both in vitro and in vivo. We describe the in vitro effect of pegylated recombinant human MGDF (PEGrHuMGDF) alone and in combination with other haemopoietic growth factors (G-CSF, GM-CSF, IL3, IL6, erythropoietin, SCF) on megakaryopoiesis in bone marrow from 11 normal subjects and 19 patients with aplastic anaemia (AA). We used semi-solid cultures to assess megakaryocyte colony growth (CFU-Mk) and 7 d suspension cultures to assess production of platelet glycoprotein IIIa (CD61) positive cells. CFU-Mk growth from normal marrow increased 3-4-fold and CD61+ve cells in suspension culture increased 8-10-fold with the addition of 10 ng/ml PEGrHuMGDF. In normal subjects growth factor combinations further increased responses in suspension culture, PEGrHuMGDF + SCF, PEGrHuMGDF + IL3 and PEGrHuMGDF + SCF + IL3 + Epo (P<0.05). IL6, GM-CSF, G-CSF or Epo added with PEGrHuMGDF did not consistently give this increase. CFU-M. growth from AA marrow remained very low in the presence of PEGrHuMGDF, with or without the addition of other growth factors. CD61+ve cells in suspension culture were, however, increased in the presence of PEGrHuMGDF alone in 12/19 AA cases. Of the 12 patients responsive to PEGrHuMGDF, nine were tested with additional growth factors and further responses were seen in six. In the AA cases PEGrHuMGDF+SCP and PEGrHuMGDF+SCF+IL3+Epo gave the highest responses. These data suggest that PEGrHuMGDF, alone or in combination with SCF and/or IL3, can enhance megakaryocyte proliferation in some patients with aplastic anaemia and may therefore have a role in the treatment of thrombocytopenia in these cases.     

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.     

In vitro growth of human fetal CD34+ cells in the presence of various combinations of recombinant cytokines under serum-free culture conditions

Summary. CD34⁺ cells were purified from midtrimester human fetal blood and adult bone marrow samples and seeded in serum-free fibrin-clot cultures in order to evaluate the number and the responsiveness to recombinant cytokines of pluripotent (CFU-GEMM), erythroid (BFU-E), megakaryocyte (BFU-meg and CFU-meg) and granulocyte/macrophage (CFU-GM) haemopoietic progenitor cells.
The number of the different haemopoietic progenitors/1 × 10³ CD34⁺ cells, except CFU-meg, was significantly higher in fetal blood than in adult bone marrow in cultures stimulated by any combination of cytokines including interleukin-3 (IL-3), granulocyte/macrophage colony stimulating factor (GM-CSF) or stem cell factor (SCF) plus erythropoietin (Epo). Nevertheless, whereas adult BFU-E showed a maximal growth in the presence of Epo plus IL-3 or Epo plus SCF, fetal BFU-E showed an optimal growth in the presence of Epo alone, the sensitivity of fetal BFU-E to suboptimal concentrations of Epo being approximately 10–15-fold higher than that of adult BFU-E. Addition of optimal concentrations of IL-3, GM-CSF or SCF, alone or in various combinations, to Epocontaining cultures induced a significant increase in both the number and size of fetal CFU-GEMM, and CFU-GM, and a parallel decrease of fetal BFU-E. Finally, SCF potently syner-gized with IL-3 in increasing the growth of both classes of fetal megakaryocyte progenitors, BFU-meg and CFU-meg.     

Porcine brain microvascular endothelial cells support the in vitro expansion of human primitive hematopoietic bone marrow progenitor cells with a high replating potential: requirement for cell-to-cell interactions and colony-stimulating factors

Primary autologous as well as allogeneic and xenogeneic stroma will support human stem cell proliferation and differentiation for several months. In the present study, we investigated the capacity of porcine microvascular endothelial cells (PMVECs) together with combinations of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] + stem factor [SCF], interleukin-3 [IL-3] + SCF + IL-6, and GM-CSF + IL-3 + SCF + IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In short-term cultures (7 days), the greatest expansion of nonadherent hematopoietic cells and clonogenic progenitors was seen with CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact), followed by PMVEC noncontact and liquid suspension cultures, respectively. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased 148.1-fold and 8.0-fold, respectively. Replating studies show that cells from day 7 dispersed blast cell colonies generated on cytokine-treated PMVEC monolayers have a high replating potential for multilineage progenitor cells. In long- term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. Maximal clonogenic cell production was observed at day 28, with 6,353- fold for total CFC and comparable increases for CFU-GM, CFU-mix, CFU- blast, BFU-E, and CFU-Mk. The total number of CD34+ cells increased 2,584-fold at day 28. Furthermore, the extended growth kinetics of these cultures indicates that these phenotypically primitive progenitor cells are also functionally expanded on PMVEC monolayers. These results support the hypothesis that direct contact with a PMVEC monolayer supports the initial expansion of hematopoietic progenitor cells with a high replating potential and, possibly, a more primitive phenotype (CD34+, CD34+/CD38-).     

FLT3/FLK2 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units

The effect of FLT3/FLK2 ligand (FL) on the growth of primitive hematopoietic cells was investigated using ThyloSca1+ stem cells. FL was observed to interact with a variety of factors to initiate colony formation by stem cells. When stem cells were stimulated in liquid culture with FL plus interleukin (IL)-3, IL-6, granulocyte colony- stimulating factor (G-CSF), or stem cell factor (SCF), cells capable of forming colonies in secondary methylcellulose cultures (CFU-c) were produced in high numbers. However, only FL plus IL-6 supported an increase in the number of cells capable of forming colonies in the spleens of irradiated mice (CFU-s). Experiments with accessory cell- depleted bone marrow (Lin- BM) showed that FL alone lacks significant colony-stimulating activity for progenitor cells. Nevertheless, FL enhanced the growth of granulocyte-macrophage progenitors (CFU-GM) in cultures containing SCF, G-CSF, IL-6, or IL-11. In these assays, FL increased the number of CFU-GM initiating colony formation (recruitment), as well as the number of cells per colony (synergy). Many of the colonies were macroscopic and contained greater than 2 x 10(4) granulocytes and macrophages. Therefore, FL appears to function as a potent costimulus for primitive cells of high proliferative potential (HPP). FL was also observed to costimulate the expansion of CFU-GM in liquid cultures of Lin- BM. In contrast, FL had no growth- promoting affects on progenitors committed to the erythrocyte, megakaryocyte, eosinophil, or mast cell lineages.     

The effect of thrombopoietin on erythroid progenitors in Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare congenital red blood cell aplasia. Usually, erythropoiesis in vitro is defective, and decreased numbers of erythroid progenitors are found in colony assays performed with bone marrow cells. Recently, some investigators showed that thrombopoietin (TPO) also works on common multipotent progenitor cells and stimulates erythropoiesis. We examined the effect of TPO together with erythropoietin (EPO), stem cell factor (SCF), and/or interleukin-3 (IL-3) on erythroid burst-forming units (BFU-E) of bone marrow nonadherent mononuclear cells from 2 patients with DBA using a serum-free culture system. Very few BFU-E appeared in cultures containing EPO alone. Adding IL-3, SCF, or both to cultures containing EPO induced an increase in the number of BFU-E. When TPO was added to cultures containing EPO and IL-3, SCF, or both, the number of BFU-E further increased. Especially in patient 2, BFU-E formation was grossly equal to that in normal controls. We conclude that TPO enhances bone marrow erythropoiesis in patients with DBA in the presence of EPO and SCF and/or IL-3. The data raise the possibility of the combination of TPO and SCF as a therapeutic agent in DBA.     

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEG rHuMGDF) on megakaryopoiesis in normal subjects and patients with myelodysplasia and acute myeloid leukaemia

Mpl ligand is a recently cloned haemopoietic growth factor that stimulates megakaryopoiesis in vitro and in vivo . We describe the in vitro effect of a truncated form of Mpl ligand, recombinant human megakaryocyte growth and development factor (rHuMGDF), on megakaryopoiesis in bone marrow from normal subjects and patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We used both semi-solid and suspension culture techniques to assess the effect of pegylated (PEG) rHuMGDF on megakaryocyte colony growth (CFU-Mk) and on the production of CD61⁺ cells in 7 d suspension cultures. PEG rHuMGDF increased CFU-Mk growth and CD61⁺ cell production in a dose-dependent fashion in all normal marrows tested. Normal CFU-Mk growth was increased threefold with the addition of 10 ng/ml PEG rHuMGDF to cultures and CD61⁺ cells were increased 8–10-fold by the same dose. Although increased CFU-Mk growth was only seen in 1/10 AML and 6/16 MDS marrows, CD61⁺ cell numbers in suspension culture were increased in 9/13 AML and 12/15 MDS samples, responses ranged from very limited to normal magnitude. There was no correlation between platelet count and CFU-Mk number, CD61⁺ cell number or response to PEG rHuMGDF. We did not find any increased CFU-GM colony or cluster growth in response to PEG rHuMGDF and the CD61⁺ cells produced in suspension culture had features of megakaryocytic differentiation. These data suggest that PEG rHuMGDF can enhance megakaryocyte proliferation in some patients with MDS and AML, and may have a role in the treatment of thrombocytopenia in these patients.     

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.     

Influence of T-lymphocytes and lactoferrin on the survival-promoting effects of IL-1 and IL-6 on human bone marrow granulocyte-macrophage and erythroid progenitor cells

Purified recombinant human (rhu) interleukin (IL)-1 alpha, rhuIL-6, iron saturated lactoferrin (LF), and T-lymphocytes were assessed for their effects on the survival of granulocyte-macrophage (granulocyte-macrophage colony-forming units, CFU-GM) and erythroid (erythroid burst-forming units, BFU-E) progenitor cells from human low-density (LD) and nonadherent LD T-lymphocyte-depleted (NALT-) bone marrow (BM) cells. Colony-stimulating factor (CSF) deprivation studies showed that 10 ng/ml IL-1 alpha could promote the survival of CFU-GM and BFU-E from NALT- BM cells. Concentrations of 1 ng/ml IL-1 alpha and 1-100 ng/ml IL-6 alone could not promote progenitor cell survival from NALT- BM cells; however, concentrations of 1 ng/ml each of IL-1 alpha and IL-6 could synergize to promote the survival of CFU-GM but not of BFU-E. The combination of these low concentrations of IL-1 alpha and IL-6 could, however, support the survival of BFU-E in the presence of purified T-lymphocytes. LF could decrease the survival of CFU-GM and BFU-E from LD but not from NALT- BM cells, apparently due to the inhibition of IL-1 release from monocytes in this cell population. The suppressive effect of LF on the survival of those progenitor cells was abolished by concentrations of 10 ng/ml IL-1 alpha or 1 ng/ml each of IL-1 alpha and IL-6. These results demonstrate that the survival of human marrow CFU-GM and BFU-E can be influenced by IL-1, IL-6, LF, and T-lymphocytes.     

Megakaryocytopoiesis in experimentally induced chronic normobaric hypoxia

It was recently proposed that prolonged hypoxia produces hypomegakaryocytic thrombocytopenia by reducing the pool of committed megakaryocyte progenitor cells at the expense of a greatly expanded erythroid progenitor pool. In order to test this hypothesis we have studied the relationship between megakaryocytopoiesis, erythropoiesis, and granulopoiesis at the level of progenitor cells (megakaryocyte colony-forming unit, CFU-Mk; erythroid CFU, CFU-E; erythroid burst-forming units; BFU-E; and granulocyte-macrophage CFU, CFU-GM) in the marrow of rats exposed for 4 weeks to normobaric hypoxia. We have found that hypomegakaryocytic thrombocytopenia was accompanied by decreased CFU-Mk, increased CFU-E, and a normal number of BFU-E and CFU-GM. These results support the hypothesis that prolonged hypoxia reduces the precursor cell commitment to differentiate into the megakaryocyte series by enhancing demand for differentiation into the erythroid cell line. However, the underlying mechanism needs further investigation.     

Basic fibroblast growth factor-stimulated ex vivo expansion of haematopoietic progenitor cells from human placental and umbilical cord blood

We investigated whether basic fibroblast growth factor (bFGF) is effective in inducing ex vivo expansion of CD34+ haematopoietic progenitor cells derived from human placental and umbilical cord blood. bFGF significantly promoted the clonal growth of various haematopoietic progenitor cells, including granulocyte-macrophage colony-forming units (CFU-GM), mixed colony-forming units (CFU-Mix) and megakaryocyte colony-forming units (CFU-Meg) under semisolid culture conditions, with an optimal bFGF concentration of 30 ng/ml. CD34+ cells were then cultured in serum-free liquid medium containing various combinations of early-acting cytokines, including thrombopoietin (TPO), stem cell factor (SCF), interleukin 3 (IL-3) and flt3-ligand (FL), with or without bFGF, for 6 and 12 d. Without bFGF, TPO + IL-3, TPO + SCF + FL and TPO +SCF + IL-3 + FL dramatically increased the total numbers of erythroid progenitors, CFU-GM and CFU-Mix by d 12 of culture respectively. However, the addition of bFGF did not promote further proliferation of these progenitors, except for the erythroid progenitors, by d 6 when stimulated with all four cytokines. In contrast, total CFU-Meg numbers were approximately doubled when these cultures were supplemented with bFGF, producing 100- to 120-fold increases compared with the baseline control cultures. These results suggest that bFGF is effective in supporting the generation of megakaryocytic progenitor cells during ex vivo expansion.     

The in vitro effects of stem cell factor, interleukin 3 and granulocyte-macrophage colony stimulating factor on haemopoietic progenitor cells from premature infants

Summary. Circulating haemopoietic progenitor cells from premature infants were assessed for their ability to respond to interleukin 3, granulocyte-macrophage colony stimulating factor and stem cell factor (SCF) in vitro. All three cytokines increased the number of colonies derived from burst forming units erythroid (BFU-E), colony forming units granulocyte-macrophage (CFU-GM) and multi-lineage progenitors (CFU-Mix) grown in the presence of erythropoietin (Epo). The size and haemoglobin content of BFU-E derived colonies also increased in the presence of the cytokines. Of those tested, SCF was found to be the most potent additive to Epo for the enhanced growth of BFU-E and CFU-Mix. In short-term liquid cultures without Epo, SCF alone induced globin synthesizing cells. Progenitors from premature infants were at least as responsive to all three cytokines as those from healthy adults. The use of SCF in combination with Epo in the prevention or treatment of anaemia in premature infants warrants further investigation.     

The in vitro effects of cytokines on expansion and migration of megakaryocyte progenitors

Increasing the number of megakaryocyte progenitors in stem cell transplants by ex vivo expansion culture may be an approach to accelerate platelet recovery in patients undergoing high-dose chemotherapy. We evaluated the effect of three different cytokine combinations on expansion, with special emphasis on the type of colony formation and migration of megakaryocytic cells. The number of clonogenic megakaryocyte progenitors (colony-forming units-megakaryocyte; CFU-Mk) with high- (> 20 cells/colony) and low-proliferative capacity (5-20 cells/colony) and the number of megakaryocytic (CD61+) cells were significantly increased by including interleukin 3 (IL-3) or IL-3 + IL-6 + IL-11 + Flt3-ligand to cultures containing megakaryocyte growth and development factor (MGDF) plus stem cell factor (SCF). No difference in the maturation of megakaryocytes from all three cytokine combinations to platelets were observed, as demonstrated by electron microscopy. In chemotaxis experiments, the migration towards stromal cell-derived factor 1 (SDF-1) was shown to be reduced for CD61+ cells and megakaryocyte progenitors cultured in other cytokines besides MGDF + SCF. The reduced migration was related to a lower expression of CXCR4, the receptor for SDF-1, on megakaryocytes from the proliferating cultures. These in vitro results demonstrate that expansion in IL-3 and other cytokines besides MGDF + SCF significantly impair the capacity of megakaryocytic cells to migrate.     

Mast cell growth factor (c-kit ligand) supports the growth of human multipotential progenitor cells with a high replating potential.

The replating capability of human multipotential (colony-forming unit-granulocyte-erythrocyte-macrophage-megakaryocyte [CFU-GEMM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitors was assessed in vitro as a potential measure of self-renewal using purified, recombinant (r) human (hu) or murine (mu) mast cell growth factor (MGF), a ligand for the c-kit proto-oncogene receptor. Primary cultures of human umbilical cord blood or adult human bone marrow cells were initiated in methylcellulose with erythropoietin (Epo) alone or in combination with rhu interleukin-3 (IL-3) or MGF. Individual day 14 to 18 CFU-GEMM or BFU-E colonies were removed from primary cultures and reseeded into secondary methylcellulose cultures containing a combination of Epo, MGF, and rhu granulocyte-macrophage colony-stimulating factor (GM-CSF). The data showed a high replating efficiency of cord blood and bone marrow CFU-GEMM in response to Epo + MGF in terms of the percentage of colonies that could be replated and the number of secondary colonies formed per replated primary colony. The average number of hematopoietic colonies and clusters apparent from replated cultures of cord blood or bone marrow CFU-GEMM stimulated by Epo + MGF was greater than with Epo + rhuIL-3 or Epo alone. Replated cord blood CFU-GEMM gave rise to CFU-GEMM, BFU-E, and GM colony-forming units (CFU-GM) in secondary cultures. Replated bone marrow CFU-GEMM gave rise mainly to CFU-GM in secondary cultures. A more limited capacity for replating of cord blood and bone marrow BFU-E was observed. These studies show that CFU-GEMM responding to MGF have an enhanced replating potential, which may be promoted by MGF. These studies also support the concept that MGF acts on more primitive progenitors than IL-3.     

Mobilization of peripheral blood progenitor cells by sequential administration of interleukin-3 and granulocyte-macrophage colony-stimulating factor following polychemotherapy with etoposide, ifosfamide, and cisplatin.

We report on the requirements that have to be met to combine a standard-dose chemotherapy regimen with broad antitumor activity with the mobilization of peripheral blood hematopoietic progenitor cells. Thirty-two cancer patients were given a 1-day course of chemotherapy consisting of etoposide (VP16), ifosfamide, and cisplatin (VIP; n = 46 cycles), followed by the combined sequential administration of recombinant human interleukin-3 (rhIL-3) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Control patients received GM-CSF alone or were treated without cytokines. Maximum numbers of peripheral blood progenitor cells (PBPC) were recruited on day 13 to 17 after chemotherapy, with a median of 418 CD34+ cells/microL blood (range, 106 to 1,841) in IL-3/GM-CSF-treated patients, 426 CD34+/microL (range, 191 to 1,380) in GM-CSF-treated patients, and 46 CD34+/microL (range, 15 to 148) in patients treated without cytokines. In parallel, there was an increase in myeloid (10,490 colony-forming unit-granulocyte-macrophage [CFU-GM]/mL blood; range, 1,000 to 23,400), as well as erythroid (10,660 burst-forming unit-erythroid [BFU-E]/mL blood; range, 3,870 to 24,300) and multipotential (840 CFU-granulocyte, erythrocyte, monocyte, megakaryocyte [GEMM]/mL blood; range, 160 to 2,070) progenitor cells in IL-3 plus GM-CSF-treated patients. In GM-CSF-treated patients, significantly less precursor cells of all lineages were mobilized, particularly multipotential progenitors (400 CFU-GEMM/mL blood; range, 200 to 2,150). Only small numbers of CD34+ cells and clonogenic progenitor cells could be recruited in intensively pretreated patients. Our data document that after standard-dose chemotherapy-induced bone marrow hypoplasia, IL-3 plus GM-CSF can be used to recruit PBPC, which might shorten the hematopoietic recovery after high-dose chemotherapy in chemosensitive lymphomas or solid tumors.     

Opposing effects of PI3 kinase pathway activation on human myeloid and erythroid progenitor cell proliferation and differentiation in vitro

OBJECTIVE: To investigate 1) the effects of lineage-specific cytokines (G-CSF and EPO) combined with ligands for different classes of cytokine receptors (common beta chain, gp130, and tyrosine kinase) on proliferation by human myeloid and erythroid progenitor cells; and 2) the signal transduction pathways associated with combinatorial cytokine actions. PATIENTS AND METHODS: CFU-GM and BFU-E were cloned in vitro. Secondary colony formation by replated CFU-GM and subcolony formation by BFU-E provided measures of progenitor cell proliferation. Studies were performed in the presence of cytokine combinations with and without signal transduction inhibitors. RESULTS: Proliferation by CFU-GM and BFU-E was enhanced synergistically when common beta chain receptor cytokines (IL-3 or GM-CSF) were combined with G-CSF or EPO, but not with gp130 receptor cytokines (LIF or IL-6) or tyrosine kinase receptor cytokines (SCF, HGF, Flt-3 ligand, or PDGF). Delayed addition studies with G-CSF+IL-3 and EPO+IL-3 demonstrated that synergy required the presence of both cytokines from the initiation of the culture. The Jak2-specific inhibitor, AG490, abrogated the effect of combining IL-3 with EPO but had no effect on the enhanced CFU-GM proliferation stimulated by IL-3+G-CSF. The PI3 kinase inhibitors LY294002 and wortmannin substituted for G-CSF in combination with IL-3 since proliferation in the presence of LY294002/wortmannin+IL-3 was enhanced to the same extent as in the presence of G-CSF+IL-3. In contrast, LY294002 and wortmannin inhibited proliferation in the presence of EPO and in the presence of EPO+IL-3. CONCLUSION: 1) IL-3 may activate different signal transduction pathways when combined with G-CSF and when combined with EPO; 2) different signal transducing intermediates regulate erythroid and myeloid progenitor cell proliferation; and 3) inhibition of the PI3 kinase pathway suppresses myeloid progenitor cell differentiation and thereby increases proliferation.     

In vivo effects of interleukin-11 and stem cell factor in combination with erythropoietin in the regulation of erythropoiesis

Summary. In this study we evaluated the in vivo effects of interleukin-11 (IL-11) and stem cell factor (SCF), in combination with erythropoietin (EPO) on murine erythropoiesis. Mice were treated for 7d with IL-11, SCF and EPO, each at three dose levels. In total, 27 different dose combinations were tested. IL-11 as well as SCF could only marginally stimulate erythroid progenitor cell numbers, but IL-11 in combination with SCF was able to increase BFU-E and CFU-E numbers 4-fold, in the absence of exogenous EPO. This resulted in an increased reticulocyte count. In contrast with the stimulatory effect on immature erythroid cell stages, IL-11 treatment induced a mild anaemia, which probably resulted from a plasma volume expansion. The additional treatment with EPO resulted in strong synergistic effects on CFU-E numbers. The combination of high-dose IL-11 and high-dose SCF was able to increase the overall efficiency of EPO-induced erythroid amplification, which was reflected by a left-shift of the in vivo EPO dose-response curve. The stimulating effects of IL-11 and SCF were further demonstrated when the effects on the reticulocyte count of a single high-dose EPO injection were assessed in normal and SCF + IL-11 treated mice. Whereas a single EPO dose increased the reticulocyte count by a factor of 3, IL-11 + SCF pretreatment increased this to a factor of 7. This study shows that in vivo SCF and IL-11 are important modulators of red blood cell production. First, these factors probably increase the input from the stem cell compartment into the erythroid lineage, where subsequently EPO is required for further amplification. Additionally, however, IL-11 and SCF increase the overall efficiency of EPO-induced amplification, probably due to a stimulatory effect on late-stage erythroid cells and to a redistribution of cells from marrow to spleen.     

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.