Regeneration of erythroid progenitor cells in polycythemic mice treated with cyclophosphamide

Mice with posthypoxic polycythemia treated with a sublethal dose of cyclophosphamide (Cy) were used as a model to investigate, by in vitro methods, the kinetics of regeneration of erythroid committed precursors (ECP) and to study the influence of erythropoietin (Ep) on those precursor cells. The results demonstrated that erythroid burst-forming units (BFU-E), early (d10) and late (d4), and erythroid colony-forming units (CFU-E) recover at different rates after Cy. Early BFU-E recovery was not Ep dependent and closely resembled regeneration of pre-erythropoietin-responsive cells (pre-ERC) found previously using the same experimental model. The absence of spontaneous recovery of mature BFU-E and CFU-E in the bone marrow and spleen of Cy-treated polycythemic mice, which is contrary to the findings in normal mice treated with Cy, indicates the importance of Ep for BFU-E (d4) and CFU-E regeneration. This was confirmed when exogenous Ep was injected. The effect on BFU-E (d4) of exogenous Ep injected into the polycythemic Cy-treated mice at the time when primitive BFU-E have regenerated considerably suggested an influence of Ep on the transition of BFU-E (d10) to BFU-E (d4). The fast regeneration of CFU-E in the spleen of normal mice and after Ep injection in polycythemic Cy-treated mice confirms the well-known and significant role of the spleen in mouse erythropoiesis under stress conditions. It could be suggested that the patterns of BFU-E (d4) and CFU-E recovery as well as Ep responsiveness closely resemble the findings observed earlier for ERC in the same experimental model.     

Biological properties of peripheral blood progenitor cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor

Patients transplanted with mobilized blood progenitor cells (PBPC) recover their neutrophil counts more rapidly than patients transplanted with bone marrow even when they receive the same dose/kg of granulocyte-macrophage colony-forming cells (CFU-GM). Here we have sought a biological explanation for this phenomenon. Most CD34-positive PBPC are quiescent (<1% in S phase) when they are collected from the bloodstream of patients treated with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF), but we have shown that they are able to resume proliferation rapidly in vitro by measuring the kinetics of CFU-GM production by primitive plastic-adherent (PΔ) cells. Also, PΔcells in PBPC harvests, unlike normal marrow PΔ cells, were insensitive to cell-cycle restraint imposed by contact with marrow-derived stromal cells. We found that PΔ cells in PBPC collections produce relatively more CFU-GM and relatively fewer BFU-E than PΔ cells in bone marrow, indicating that granulopoiesis might occur at the expense of erythropoiesis, but we were unable to find any differences in the kinetics of granulocytic maturation between PBPC and bone marrow. Our interpretation of these findings is that transplanted PBPC rapidly enter the cell cycle and contact with stromal cells in the marrow does not reduce the proportion of progenitors participating in neutrophil production. Consequently, neutrophil recovery after PBPC infusion is more rapid than neutrophil recovery after marrow infusion. Granulopoiesis at the expense of erythropoiesis may also contribute to this effect.     

The cell surface form of colony-stimulating factor-1 is biologically active in bone in vivo

The specific biological function of the cell surface or membrane-bound isoform of colony-stimulating factor-1 (mCSF-1) is not well understood. To help define the role of this isoform in bone, we developed a transgenic mouse in which targeted expression of human mCSF-1 in osteoblasts was achieved under the control of the 2.4-kb rat collagen type I alpha promoter. Bone density, determined by peripheral quantitative computed tomography, was reduced 7% in mCSF-1 transgenic compared with that in wild-type mice. Histomorphometric analyses indicated that the number of osteoclasts in bone (NOc/BPm, NOc/TAR, OcS/BS) was significantly increased in transgenic mice (1.7- to 1.8-fold; P < 0.05 to P < 0.01) compared with that in wild-type animals. Interestingly, the osteoblast-restricted isoform transgene corrected the osteopetrosis seen in CSF-1-deficient op/op mice. Skeletal growth and bone density in op/op mice expressing mCSF-1 in osteoblasts were similar to those in wild-type mice and were dramatically different from those in the unmanipulated op/op animals. The op/op mice expressing mCSF-1 in bone had normal incisor and molar tooth eruption, whereas the op/op mice evidenced the expected failure of tooth eruption. These findings directly support the conclusion that mCSF-1 is functionally active in bone in vivo and is probably an important local source of CSF-1.     

Abnormal responses of myeloid progenitor cells to recombinant human colony-stimulating factors in congenital neutropenia.

The effects of recombinant human interleukin-3 (IL-3) and recombinant human granulocyte colony-stimulating factor (G-CSF) on the growth of myeloid progenitor cells (CFU-C) in semisolid agar culture were studied in two patients with Kostmann-type congenital neutropenia. CFU-C growth in bone marrow cells from patients was significantly reduced in response to various concentrations of either IL-3 or G-CSF alone, compared with that from normal subjects. There was no inhibitory effect of bone marrow cells from patients on normal CFU-C formation supported by IL-3 or G-CSF. However, the simultaneous stimulation with IL-3 and G-CSF induced the increase of CFU-C formation in patients with congenital neutropenia. Furthermore, CFU-C growth in both patients was supported when bone marrow cells were preincubated with IL-3 in liquid culture followed by the stimulation with G-CSF in semisolid agar culture. In contrast, that was not supported by the preincubation with G-CSF and the subsequent stimulation with IL-3. This evidence suggests that the hematopoietic progenitor cells in patients with congenital neutropenia have the potential for developing CFU-C in the combined stimulation with IL-3 and G-CSF, and that this growth may be dependent on the priming of IL-3 followed by the stimulation with G-CSF. The level of mature neutrophils in peripheral blood was not fully restored to normal levels by the daily administration of G-CSF in doses of 100 to 200 micrograms/m2 of body surface area for 20 to 25 days in both patients. These observations raise the possibility that the combination of IL-3 and G-CSF might have a potential role for the increase of neutrophil counts in patients with congenital neutropenia.     

Insulin-like growth factor-1 short-period therapy improves cardiomyopathy stimulating cardiac progenitor cells survival in obese mice

Background and aimsCardiovascular diseases are the main cause of mortality in obesity. Despite advanced understanding, the mechanisms that regulate cardiac progenitor cells (CPC) survival in pathological conditions are not clear. Low IGF-1 plasma levels are correlated to obesity, cardiomyopathy and CPC death, so this work aimed to investigate IGF-1 therapeutic potential on cardiomyopathy and its relationship with the survival, proliferation and differentiation of CPC in Western diet-induced obesity.Methods and resultsMale Swiss mice were divided into control group (CG, n = 8), fed with standard diet; and obese group (OG, n = 16), fed with Western diet, for 12 weeks. At 11th week, OG was subdivided to receive a daily subcutaneous injection of human recombinant IGF-1 (100 μg.Kg⁻¹) for seven consecutive days (OG + IGF1, n = 8). Results showed that IGF-1 therapy improved the metabolic parameters negatively impacted by western diet in OG, reaching levels similar to CG. OG + IGF-1 also demonstrated restored heart energetic metabolism, fibrosis resolution, decreased apoptosis level, restored cardiac gap junctions and intracellular calcium balance. Cardiomyopathy improvement was accompanied by increased CPC survival, proliferation and newly cardiomyocytes formation related to increased pAkt/Akt ratio.ConclusionThese results suggest that only one week of IGF-1 therapy has cardioprotective effects through Akt pathway upregulation, ensuring CPC survival and differentiation, contributing to heart failure rescue.     

The activity in ex vivo expansion of cord blood myeloid progenitor cells before and after cryopreservation

A total of 50 human umbilical cord blood (UCB) samples were studied. The hematopoietic stem/progenitor (CD34+) populations were isolated from UCB mononuclear cells (MNC) by means of immunomagnetic separation. Double immunofluorescent staining of UCB CD34+ cells revealed that there was a high proportion (82.33 +/- 4.47%) of CD34+ cells co-expressing CD13, while the percentage of CD34+ CD33+ cells was much lower (22.17 +/- 3.35%). In contrast, for co-expressing lymphoid differentiation antigens, the proportion of CD34+CD38+ cells (38.34 +/- 6.09%) was relatively higher than that of CD34+CD10+ cells (11.52 +/- 1.24%) or CD34+CD2+ cells (9.84 +/- 2.30%). For stimulating the ex vivo expansion of UCB progenitor cells, no single hematopoietic growth factor (HGF) was efficacious when used alone, while combination of 4 HGFs, such as GM-CSF, G-CSF, IL-3, and SCF could induce a 55-fold increase in the myeloid progenitor cells, day-14 CFU-GM, in a short term of 7 days' liquid culture. Cryopreservation of UCB as MNC preparations at -196 degrees C could satisfactorily retain the number and activity of CD34+ cells. After thawing, a high recovery rate of about 80% CD34+ cells was obtained. When suspended in liquid cultures containing a combination of 4 HGFs, as shown above, the frozen cord blood progenitor cells could be well expanded, reaching a >50-fold increase in day-14 CFU-GM, which was very similar to that of the fresh UCB samples. In addition, a similar result was also seen in CFU-GEMM, indicating that after cryopreservation the recovered UCB progenitor cells retain an intact clonogeneic ability capable of efficiently responding to hematopoietic growth factors for ex vivo expansion.     

Abnormalities of primitive myeloid progenitor cells expressing granulocyte colony-stimulating factor receptor in patients with severe congenital neutropenia

To define the basis for faulty granulopoiesis in patients with severe congenital neutropenia (SCN), the expression of granulocyte colony-stimulating factor receptor (G-CSFR) in primitive myeloid progenitor cells and their responsiveness to hematopoietic factors were studied. Flow cytometric analysis of bone marrow cells based on the expression of CD34, Kit receptor, and G-CSFR demonstrated a reduced frequency of CD34(+)/Kit(+)/ G-CSFR(+) cells in patients with SCN. The granulocyte-macrophage colony formation of CD34(+)/Kit(+)/G-CSFR(+) cells in patients was markedly decreased in response to G-CSF alone and to the combination of stem cell factor, the ligand for flk2/flt3, and IL-3 with or without G-CSF in serum-deprived semisolid culture. In contrast, no difference in the responsiveness of CD34(+)/Kit(+)/G-CSFR(-) cells was noted between patients with SCN and subjects without SCN. These results demonstrate that the presence of qualitative and quantitative abnormalities of primitive myeloid progenitor cells expressing G-CSFR may play an important role in the impairment of granulopoiesis in patients with SCN. (Blood. 2000;96:4366-4369)     

Synergistic myelopoietic actions in vivo after administration to mice of combinations of purified natural murine colony-stimulating factor 1, recombinant murine interleukin 3, and recombinant murine granulocyte/macrophage colony-stimulating factor.

Combinations of low dosages of purified murine hematopoietic colony-stimulating factors (CSFs)--L-cell CSF type 1 (CSF-1), recombinant interleukin 3 (IL-3), and recombinant granulocyte/macrophage CSF (GM-CSF)--were compared with single CSFs for their influence on the cycling rates and numbers of bone marrow granulocyte/macrophage, erythroid, and multipotential progenitor cells in vivo in mice pretreated with human lactoferrin. Lactoferrin was used to enhance detection of the stimulating effects of exogenously administered CSFs. Concentrations of CSFs that were not active in vivo when given alone were active when administered together with other types of CSF. The concentrations of CSF-1, IL-3, and GM-CSF needed to increase progenitor cell cycling rates were reduced by factors of 40-200, 10-50, and 40- greater than 400, respectively; the concentrations needed to increase progenitor cell numbers were reduced by factors of 40-500 (CSF-1), 20-80 (IL-3), and greater than 40- greater than 200 (GM-CSF) when these forms of CSFs were administered in combination with low dosages of one of the other forms of CSFs. The results demonstrate that different CSFs can synergize when administered in vivo to increase the cycling rates and numbers of marrow hematopoietic progenitor cells. These findings may be of relevance physiologically to the regulation of myeloid blood cell production by CSFs.     

Induction of proliferation of purified human myeloid progenitor cells: a rapid assay for granulocyte colony-stimulating factors

The proliferation and differentiation of granulocyte and monocyte progenitor cells (CFU-C) in vitro is dependent on the presence of a group of closely related glycoproteins termed colony-stimulating factors (CSF). In order to investigate the interaction of these factors with CFU-C, we purified CFU-C from the peripheral blood of chronic myeloid leukemia patients with an immune rosette technique using specific monoclonal antibodies (mean 74-fold enrichment, 45% cloning efficiency). Colony formation by purified CFU-C demonstrated an absolute dependence on an exogenous source of CSF. Liquid culture of small aliquots of enriched CFU-C with CSF-containing medium resulted in a rapid, time- and concentration-dependent induction of DNA synthesis as measured by 3H-thymidine incorporation. This specific CSF induction of DNA synthesis by enriched CFU-C was used to develop a microassay system for CSF activity. CSF activity could be reproducibly quantitated in 24-48 hr. The proliferating cells in this assay system were shown to be myeloid progenitor cells by examining the morphology of their progeny and by determining the surface antigen phenotype of the responding cells (Ia+, T3-, B1-, Mo1-). This microassay provides a quantitative assessment of CSF activity that may be useful in the purification of human CSF and in the generation of monoclonal antibodies to CFU-C surface structures.     

Sources of murine macrophage colony-stimulating factor that also contain growth factors for primitive macrophage progenitor cells

A new growth factor (synergistic factor, SF) has recently been described, which, in combination with a macrophage CSF source, is able to stimulate the proliferation of primitive high-proliferative-potential macrophage-progenitor cells (HPP-CFC) in mouse bone marrow in culture. It has been found that the addition of supraoptimal amounts of the crude CSF sources, extracts of pregnant mouse uterus and embryo, media conditioned by L cells or the mouse mammary tumor cell line (EMT6), stimulated the proliferation of HPP-CFC in the absence of any added SF. These preparations thus appear to contain a factor with similar biological properties to those of SF. This conclusion is supported by the results obtained from Sephacryl S200 chromatography of these three CSF sources, which indicate that in all three cases fractions with apparent molecular weight (MW) greater than 68,000 contained the major portion of the CSF-1 activity, whereas fractions with MW less than 68,000 contained the SF-like activity, together with minor amounts of CSF-1 activity.     

Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor

Committed progenitor cells and primitive stem cells mediate early and sustained engraftment, respectively, after lethal irradiation and stem cell transplantation. Peripheral blood stem cells (PBSC) from unstimulated mice are deficient in both cell types. To study techniques to mobilize both progenitor cells and primitive stem cells from the marrow to the blood, we collected peripheral blood from C57BL/6 mice 6 to 7 days after a single dose of cyclophosphamide (CY; 200 mg/kg intraperitoneally), after recombinant human granulocyte colony- stimulating factor (rhG-CSF) (250 micrograms/kg/d twice per day subcutaneously for 4 days), or after CY followed by G-CSF. Significant increases in white blood cell counts (1.6- to 2.7-fold) and circulating day 8 colony-forming unit spleen (CFU-S) (11- to 36-fold) were seen with all three mobilization methods compared with unstimulated control mice. Transplantation of mobilized blood stem cells into lethally irradiated hosts decreased the time to erythroid engraftment. Blood stem cells were analyzed for primitive stem cell content by Rs, an assay for CFU-S self-renewal, and competitive repopulation index (CRI), an assay of long-term repopulating ability. The primitive stem cell content of unstimulated blood was clearly deficient, but was significantly increased following mobilization, approaching normal bone marrow levels. These results were confirmed by an in vitro limiting dilution long-term culture assay that measures the frequency of progenitor cells and primitive stem cells. Mobilization following CY + G-CSF was accompanied by a marked loss of both progenitor cells and primitive stem cells in the marrow. In contrast, following G-CSF alone the progenitor cell and primitive stem cell content of the marrow was unchanged. Stem cell mobilization following CY + G-CSF was not affected by previous exposure of donors to cytosine arabinoside or cyclophosphamide, but was significantly reduced by previous exposure to busulfan. These data show that stem cell content in the blood may reach near-normal marrow levels after mobilization, the mobilization from the marrow to the blood is temporary and reversible, the specific technique used may mobilize different subpopulations of stem cells, and the type of prior chemotherapy may influence the ability to mobilize stem cells into the blood.     

The role of interleukin-1 and granulocyte-macrophage colony-stimulating factor in the paracrine stimulation of an in vivo-derived murine myeloid leukemia

WEHI-274.3 is a cell line isolated from an in vivo-derived, murine myelomonocytic leukemia. Although the survival and growth of WEHI-274.3 cells in vitro is absolutely dependent on the addition of exogenous growth factors such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or colony-stimulating factor-1, when injected into syngeneic mice the cell line is tumorigenic. Sera from normal mice contain low levels of an activity that sustains survival of WEHI-274.3 but does not stimulate growth. In contrast, sera from mice bearing the WEHI-274.3 leukemia contained levels of CSF-1 and GM-CSF that stimulated the growth of WEHI-274.3 cells. Supernatants of cultures of WEHI-274.3 cells contained an activity that stimulated 3T3 fibroblasts to release an activity that stimulated the growth of the WEHI-274.3 cells. The 3T3-stimulatory activity released by the WEHI-274.3 cells was neutralized completely with an antiserum specific for murine IL-1 alpha, but not with antiserum specific for IL-1 beta. Moreover, WEHI-274.3 cells both in vitro and in vivo contained high levels of IL-1 alpha and IL-1 beta mRNAs. The leukemia-stimulatory activity released by the 3T3 cells was neutralized by an antiserum specific for GM-CSF. We postulate that the IL-1 alpha constitutively released by the WEHI-274.3 cells stimulates the production of GM-CSF from host cells such as fibroblasts or endothelial cells. A similar paracrine mechanism of growth stimulation may occur in acute myeloid leukemias in humans.     

Temporal response of murine pluripotent stem cells and myeloid and erythroid progenitor cells to low-dose glucan treatment

B6D2F1 female mice were intravenously administered 0.4 mg of glucan. 1, 5, 11, and 17 days later, the total nucleated cellularity (TNC) and the numbers of pluripotent hemopoietic stem cells (CFU-s), granulocyte-macrophage progenitor cells (GM-CFC), and erythroid colony-forming (CFU-e) and burst-forming (BFU-e) cells were assayed in the bone marrow and spleen. Bone marrow TNC was not altered, but splenic TNC increased approximately twofold on day 5 and remained increased on days 11 and 17 after glucan treatment. The concentrations of bone marrow and splenic CFU-s and GM-CFC both significantly increased (p less than 0.01) by 5 days after glucan administration; however, they returned to control levels by day 17. Splenic CFU-e concentration increased on days 5, 11, and 17, whereas splenic BFU-e concentration increased only on day 11 after treatment. By contrast, bone marrow CFU-e and BFU-e concentrations were either unaffected or slightly decreased by glucan treatment. When peripheral blood was assayed for CFU-s and GM-CFC, no detectable increase in the concentrations of these progenitors was noted at any time after glucan treatment. The relevance of these effects of low-dose (0.4 mg) glucan treatment is discussed with respect to previously reported effects of higher-dose (e.g., 4.0 mg) glucan treatment.     

Factors influencing collection of peripheral blood progenitor cells following high-dose cyclophosphamide and granulocyte colony-stimulating factor in patients with multiple myeloma

We treated 103 multiple myeloma (MM) patients with 7 g/m² cyclophosphamide (Cy) followed by 300 μg G-CSF/d to harvest peripheral blood progenitor cells (PBPC). PBPC autografts containing > 2.0 × 10⁶ CD34⁺ cells per kg body weight were obtained at the first attempt from 90/100 evaluable patients. The most significant factor predicting impairment of PBPC collection was the duration of previous melphalan treatment ( P  < 0.0001). In multivariate discriminate analysis, treatment with melphalan during the most recent chemotherapy cycles prior to mobilization ( P  = 0.0727) and previous radiotherapy ( P  = 0.0628) had a marginally significant negative influence on the efficacy of PBPC collection. We found no reduced functional capacity of CD34⁺ cells to restore haemopoiesis after myeloablative treatment related to the duration of melphalan exposure. At the time of best response to conventional treatment, a median paraprotein reduction of 21% was achieved following high-dose cyclophosphamide (HD-Cy). Two heavily pretreated patients died and one patient developed pulmonary toxicity W.H.O. grade IV following HD-Cy. Potential transplant candidates should undergo mobilization and harvesting of PBPC before melphalan-containing treatment. Combinations of haemopoietic growth factors and their dose modifications should be investigated to improve PBPC collection, to allow a dosage reduction of the mobilization chemotherapy.     

Effects of B cell stimulatory factor-1/interleukin 4 on hematopoietic progenitor cells

B cell stimulatory factor-1 (BSF-1)/Interleukin 4 (IL 4) is a T cell product originally characterized on the basis of its actions on B lymphocytes. Recently it has been reported that BSF-1 activates T cell and mast cell lines. We now provide evidence that BSF-1, purified to homogeneity, also has a broad spectrum of activity on hematopoietic progenitor cells (HPC). However, like its action on B cells, prolierative effects were only observed when BSF-1 was combined with an additional factor. Thus BSF-1, in costimulation with recombinant G-CSF, enhances the proliferation of granulocyte-macrophage progenitor cells (CFU-GM). BSF-1 increases the proliferation of CFU-e in the presence of recombinant erythropoietin (rEPO). Furthermore, BSF-1 induces, together with rEPO, colony formation by primitive erythroid (BFU-e) and multipotent (CFU-mix) progenitor cells comparable to that observed with rEPO and interleukin 3 (IL 3). BSF-1 is also active as a megakaryocyte colony-stimulating factor; in combination with recombinant interleukin 1, rEPO or the supernatant of the T cell hybridoma FS7-20.6.18, BSF-1 induces megakaryocyte colony formation (CFU-Mk). The same factors that synergize with BSF-1 also enhance CFU-Mk proliferation induced by IL 3. Although the precise mechanisms of action of BSF-1 on HPC is not yet known, we propose that BSF-1 represents an activation factor for HPC and prepares the progenitor cells to respond to specific growth or differentiation factors.