Marrow Progenitor Cells Research Articles

Regulation of Platelet Production and Function by Megakaryocyte Growth and Development Factor in Nonhuman Primates

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu-MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5-fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating concentration of platelets for both EA and VG (r=.98 in both cases), without significant changes in fibrin accumulation (P > .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1-6) (P < .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P > .5), rHu-MGDF enhanced aggregatory responses to low doses of physiologic agonists, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P-selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.

Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu- MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P &lt; 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P &lt; 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P &lt; 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P &gt; .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P &lt; 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P &lt; 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P &lt; 10(-4)), resulting in a 6.5- fold increase in marrow megakaryocyte mass (P &lt; 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating concentration of platelets for both EA and VG (r=.98 in both cases), without significant changes in fibrin accumulation (P &gt; .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1–6) (P &lt; .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P &gt; .5), rHu-MGDF enhanced aggregatory responses to low doses of physiologic agonists, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P- selectin, or Annexin V binding sites; P &gt; .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.

Role of c-myc in myeloid differentiation, growth arrest and apoptosis.

Hematopoiesis is a profound example of cell homeostasis which is regulated throughout life, whereby a hierarchy of hematopoietic progenitor cells in the bone marrow (BM) proliferate and terminally differentiate along multiple, distinct cell lineages. This includes the proliferation and differentiation of myeloid precursor cells into granulocytes and macrophages (33, 40, 42). Clearly, a variety of control mechanisms are needed to maintain steady state levels of mature blood cells, as well as to stimulate the rapid production of specific cell types as needed. To achieve this requires the participation of many factors, including positive and negative regulators of growth and differentiation, which determine survival, growth stimulation, differentiation, functional activation, and programmed cell death (apoptosis) (41, 44).

The effects of hyperthermia in bone marrow purging of breast cancer.

The number of autologous bone marrow transplants done for solid tumours, particularly breast cancer, has risen steadily over the last ten years. The role of bone marrow or peripheral blood progenitor cell purging in transplantation is incompletely understood. Theoretically, the reinfusion of untreated bone marrow containing tumour cells might result in relapse in some patients treated with high-dose chemotherapy and hematopoietic support. Therefore, safe and effective purging techniques may increase long-term, disease-free survivorship. In this study, hyperthermia was evaluated for its ability to purge CAMA-1 breast cancer cells from normal human bone marrow. Between two and nine trials of a range of temperatures (42-45 degrees C) and durations of treatment (1-4 h) were performed. The effect of hyperthermia on normal bone marrow alone and in mixes with breast cancer cells was also evaluated. Hyperthermia (45 degrees C, 4 h) produced > 5 logs of CAMA-1 cell kill. Exposures of 45 degrees C for 2 h and 44 degrees C for 4 h resulted in approximately three logs of cell kill, corresponding to < 1% survival of clonogenic cells. Normal bone marrow was considerably more vulnerable to heat treatments, however, with approximately 1% of progenitors remaining clonogenic after exposure of 43 degrees C for 2 h and 44 degrees C for 1 h. Therefore, although hyperthermia is able to achieve adequate CAMA-1 breast cancer cell kill, it remains more toxic to normal bone marrow as a purging method. To make hyperthermia useful in purging systems, mechanisms to selectively alter thermal sensitivity must be pursued.

Combined Use of Growth Factors to Stimulate the Proliferation of Hematopoietic Progenitor Cells after Autologous Bone Marrow Transplantation for Lymphoma Patients

We studied the kinetic response and concentration of bone marrow (BM) progenitor cells of patients with lymphoid malignancies submitted to autologous bone marrow transplantation (ABMT), treated with a granulocyte-colony-stimulating factor (G-CSF)/interleukin-3 (IL-3) combination. The results were compared with those of lymphoma patients receiving the same pretransplant conditioning regimen followed by G-CSF alone. Recombinant human (rh)G-CSF was administered as a single subcutaneous (s.c.) injection at the dose of 5 micrograms/kg/day from day + 1 after reinfusion of autologous stem cells, while rhIL-3 was added from day +6 at the dose of 10 micrograms/kg/day s.c. (overlapping schedule). In both groups (i.e. G-CSF- and G-CSF/IL-3-treated patients), cytokine administration was discontinued when the absolute neutrophil count was > 0.5 x 10(9)/l of peripheral blood for 3 consecutive days. Following treatment with the CSF combination, the percentage of marrow CFU-GM and erythroid progenitors (BFU-E) in the S phase of the cell cycle increased from 9.3 +/- 2 to 33.3 +/- 12% and from 14.6 +/- 3 to 35 +/- 6%, respectively (p < 0.05). The number of actively cycling megakaryocyte progenitors (CFU-MK and BFU-MK) also increased. Conversely, G-CSF augmented the proliferative rate of CFU-GM (22.6 +/- 6% compared to a baseline value of 11.5 +/- 3%; p < 0.05) but not of BFU-E, CFU-MK or BFU-MK, and the increase in S-phase CFU-GM was significantly lower than that observed in the posttreatment samples of patients receiving IL-3 in addition to G-CSF. The absolute number of both CFU-GM and BFU-E/ml of BM was significantly augmented after treatment with G-CSF/IL-3 but not G-CSF alone. Similarly, administration of the cytokine combination resulted in a higher number of CD34+ cells and their concentration was significantly greater than that observed in the posttreatment samples of G-CSF patients. We also investigated the responsiveness to CSFs, in vitro, of highly enriched CD34+ cells, collected after priming with G-CSF in vivo (i.e. after 5 days of G-CSF administration). Our results demonstrated that pretreatment with G-CSF modified the response of BM cells to subsequent stimulation with additional CSFs. When the hematological reconstitution of patients treated with G-CSF/ IL-3 was compared to that of individuals receiving G-CSF alone, the addition of IL-3 resulted in a significant improvement in granulocyte and platelet recovery, a lower transfusion requirement and shorted hospitalization. In conclusion, our results indicate that in vivo administration of two cytokines increases the proliferative our results indicate that in vivo administration of two cytokines increase the proliferative our results rate and concentration of BM progenitor cells better than G-CSF alone and support a role for growth factor combinations for accelerating hematopoietic recovery after high-dose chemotherapy.

Autologous Progenitor Cell Transplantation: Prior Exposure to Stem Cell-Toxic Drugs Determines Yield and Engraftment of Peripheral Blood Progenitor Cell But Not of Bone Marrow Grafts

Agents with stem cell-toxic potential are frequently used for salvage therapy of Hodgkin's disease (HD) and high-grade non-Hodgkin's lymphoma (NHL). Because many patients with relapsed or refractory lymphoma are candidates for autologous progenitor cell transplantation, possible toxic effects of salvage chemotherapy on progenitor cells must be taken into account. In a retrospective study, we have analyzed the influence of a salvage regimen containing the stem cell-toxic drugs BCNU and melphalan (Dexa-BEAM) on subsequently harvested bone marrow (BM)- and peripheral blood-derived progenitor cell grafts (PBPC) and compared it with other factors. Progenitor cells were collected from 96 patients with HD or high-grade NHL. Seventy-nine grafts were reinfused (35 PBPC and 44 BM) after high-dose chemotherapy. Compared with patients autografted with BM, hematopoietic recovery was significantly accelerated in recipients of PBPC. For PBPC, the number of Dexa-BEAM cycles ( > or = v > 1) was the predominate prognostic factor affecting colony-forming unit-granulocyte-macrophage (CFU-GM) yield (66 v 6.8 x 10(4)/kg, P = .0001), CD34+ cell yield (6.6 v 1.6 x 10(6)/kg, P = .0001), neutrophil recovery to > 0.5 x 10(9)/L (9 v. 11 days, P = .0086), platelet recovery to > 20 x 10(9)/L (10 v 15.5 days, P = .0002), and platelet count on day +100 after transplantation (190 v 107 x 10(9)/L, P = .031) using univariate analysis. Previous radiotherapy was associated with significantly lower CFU-GM and CD34+ cell yields but had no influence on engraftment. Patient age, patient sex, disease activity, or chemotherapy other than Dexa-BEAM did not have any prognostic impact. Multivariate analysis confirmed that Dexa-BEAM chemotherapy was the overriding factor adversely influencing CFU-GM yield (P < .0001), CD34+ cell yield (P < .0001), and platelet engraftment (P < .0001). BM grafts were not significantly affected by previous Dexa-BEAM chemotherapy or any other variable tested. However, prognostic factors favoring the use of BM instead of PBPC were not identified using joint regression models involving interaction terms between the graft type (PBPC or BM) and the explanatory variables investigated. We conclude that, in contrast to previous radiotherapy or other chemotherapy, exposure to salvage regimens containing stem cell-toxic drugs, such as BCNU and melphalan, is a critical factor adversely affecting yields and performance of PBPC grafts. Marrow progenitor cells appear to be less sensitive to stem cell-toxic chemotherapy. PBPC should be harvested before repeated courses of salvage chemotherapy involving stem cell-toxic drugs to preserve the favorable repopulation kinetics of PBPC in comparison with BM.

1118 Amifostine improves the antileukemic therapeutic index of mafosfamide: Implications for bone marrow purging

To test the potential use of amifostine (Ami) to protect normal bone marrow (N1 BM) progenitor cells during ex vivo purging of leukemia, the dose response of mafosfamide (Mfs) ± Ami on N1 BM progenitor cells was determined and the LD95 was calculated. Ami pretreatment resulted in a statistically significant protection of CFU-GM and erythroid blast-forming units (BFU-E) from Mfs toxicity. The mean ± SEM μg/ml LD95 concentrations were: CFU-GM, Mfs alone 54 ± 13 vs Ami-Mfs 66 ± 14, p. 005, BFU-E, Mfs alone 48 ± 14, vs Ami-Mfs 61 ± 11, p .005. In contrast, Ami pretreatment sensitized CFU-L to Mfs toxicity: LD95 for Mfs alone 33 ± 4 vs Ami-Mfs 27 ± 3, p .003. At the LD95 fur CFU-GM and BFU-E, Mfs alone resulted in a ≈4 log cell-kill for L-CFU. Ami protection of N1 BM resulting in a higher Mfs LD95 concentration, coupled with the Ami-sensitization of the leukemia, provided an estimated additional 6+ log leukemia cell-kill. This enhanced selective leukemia cell-kill from Mfs during ex vivo purging using the LD95 concentration of Mfs for normal marrow progenitors offers the potential for lowering the incidence of leukemic relapse while preserving a greater number of normal progenitor cells for engraftment following autologous transplantation.

Proliferation and differentiation of myelodysplastic CD34+ cells in serum-free medium: II. Response to combined colony-stimulating factors.

To investigate the role of colony stimulating factors (CSFs) in the proliferation and differentiation of progenitor cells from myelodysplastic syndromes (MDS), marrow progenitor cells from 18 MDS patients were highly purified using CD34 monoclonal antibody and immunomagnetic microspheres (MDS CD34+ cells). These cells were cultured in serum-free medium with various combinations of five colony stimulating factors (CSFs): recombinant human interleukin-3 (rIL-3), granulocyte/macrophage-CSF (rGM-CSF), granulocyte-CSF (rG-CSF), macrophage-CSF (rM-CSF), and erythropoietin (rEP). Among the tested CSFs, such as rM-CSF, rG-CSF, rGM-CSF and rIL-3, a combination of the first three CSFs was the most effective stimulus for the proliferation of non-erythroid MDS progenitor cells. An increase of undifferentiated "blast" cell colonies in 5/18 MDS patients occurred and these 5 patients belonged to the high-risk group. In the presence of these three CSFs, rIL-3 had no effect on the proliferation and differentiation of MDS CD34+ cells; however, IL-3 was efficient for the proliferation of MDS CD34+ cells to the erythroid lineage. rGM-CSF or rIL-3 alone did not efficiently support proliferation and differentiation of CD34+ cells. M-CSF is present in normal human serum at a concentration of 550 +/- 110 U/ml, a concentration exceeding that used in this study (100 U/ml). Therefore, in vivo administration of G-CSF combined with GM-CSF to MDS patients may be one of the most effective CSF combinations for proliferation of MDS progenitor cells to the non-erythroid lineage. However, the effect on the capacity for differentiation was minimal, especially in patients belonging to the high-risk group.

Effects of Cytokines on Expansion and Maintenance of Hematopoietic Progenitor Cells of Bone Marrow Obtained from Children with Solid Tumors and Acute Leukemias

Effects of Cytokines on Expansion and Maintenance of Hematopoietic Progenitor Cells of Bone Marrow Obtained from Children with Solid Tumors and Acute Leukemias

A comparison of two GM-CSF schedules to counteract the granulo-monocytopenia of carboplatin-etoposide chemotherapy

In order to obtain the beneficial effects from granulocyte-macrophage colony-stimulating factor (GM-CSF) on granulo-monocyte recovery with the minimum dose and toxicity, we compared the effect of two different GM-CSF schedules (5 micrograms/kg/day subcutaneously, days 5 to > 18 versus days 12 to > 18 on the cytopenias which follow cytostatic treatment with carboplatin (400 mg/m2 intravenous (i.v.) day 1) and etoposide (100 mg/m2 i.v. days 1 to > 3). 13 patients entered the study for a total of 36 evaluable cycles. The cytostatic treatment produced a neutropenia that persisted for up to day 22 (absolute neutrophil count (ANC) < 1000/microliters in 25% and ANC < 2000 in 50% of control cycles). Early GM-CSF administration markedly increased the leucocyte nadir and produced two waves of leucocytosis: an early one, linked to marrow reserve release and presumably of no value to the patients; and a delayed one, due to marrow precursor and progenitor cell proliferation, in which the granulomonocytosis was associated with a marked eosinophilia. The delayed GM-CSF administration markedly increased the leucocyte nadir and accelerated granulo-monocyte recovery (with an only modest eosinophilia), so that chemotherapy could be repeated every 21 days in all the patients.

Gene transfer into human hemopoietic progenitor cells

Considerable progress is being made in the transfer of genetic material to hematopoietic stem cells. In this chapter we describe how gene transfer is being used to: mark marrow and peripheral blood progenitor cells prior to autologous transplantation, to track their fate on reinfusion and to detect contaminating tumorigenic cells; modulate immunocyte function--important in immunologic disorders and perhaps in cancer therapy; generate tumor vaccines from tumor cells isolated from marrow; correct single gene defects--the 'classical' concept of gene therapy; and finally to modify the drug sensitivity of progenitor cells--enabling them to resist the suppressive effects of cytotoxic drugs during cancer therapy and perhaps providing a mechanism for in vivo selection of gene modified cells.

Immunologically Based Methods for the Elimination of Tumor Cells from Autologous Stem Cell Grafts

There is currently renewed interest in the potential of tumor cells within autologous hematopoietic grafts to contribute to relapse of cancer post-transplant. This has prompted the development of a wide variety of techniques for the ex vivo removal or purging of malignant cells from bone marrow and peripheral blood progenitor cell grafts. Both negative selection, in which tumor cells are eliminated, and positive selection, in which hematopoietic stem cells are enriched, are under examination as purging modalities. This review describes the use of immunologically based methods for autologous graft engineering.

The role and methodology for purging tumor from autologous bone marrow and peripheral blood progenitor cells.

The role and methodology for purging tumor from autologous bone marrow and peripheral blood progenitor cells.

Limited value of myeloablative therapy for late multiple myeloma

The utility of myeloablative therapy supported by autologous bone marrow (BM) or blood progenitor cells was assessed in 49 patients with multiple myeloma who had received at least 1 year of prior chemotherapy. Outcomes were compared with those of similar patients who did not receive intensive treatment primarily for socioeconomic reasons. Among patients with disease in resistant relapse despite treatment with vincristine-doxorubicin by continuous infusion with pulse dexamethasone (VAD), a 61% response rate was associated with a median remission time of 5 months. After primary resistance for more than 1 year, 6 of 15 patients responded and the overall survival was similar to that of control patients. For patients with melphalan- resistant disease that responded to VAD, the remission time was similar to that of control patients. Current myeloablative treatments supported by autologous BM or blood stem cells were useful to very few patients with multiple myeloma after the first year of chemotherapy.

▾ Filgrastim – human granulocyte-colony stimulating factor

Filgrastim (recombinant human granulocyte-colony stimulating factor) (Neupogen – Amgen-Roche) was the first of several white cell ‘colony stimulating factors’ to be licensed for use in Britain. Filgrastim stimulates specifically the production of neutrophils from progenitor cells in the bone marrow, speeds their maturation and increases their number in the peripheral blood.1,2 Use of filgrastim is intended to reduce the duration and severity of myelosuppression that follows intensive cytotoxic chemotherapy. The manufacturer claims that filgrastim “supports full dose chemotherapy on time”, by moderating neutropenia and reducing the risk of infection that might interfere with the next cycle of treatment. When should filgrastim be given?

Proliferation and differentiation of myelodysplastic CD34+ cells in serum‐free medium: response to individual colony‐stimulating factors

The presence of serum or contaminant cells may alter clonal development of haematopoietic progenitor cells in vitro. To investigate the pathogenesis of myelodysplastic syndromes (MDS), marrow progenitor cells from 13 MDS patients were highly purified using monoclonal antibodies including CD34 and immunomagnetic microspheres. The cells positive for CD34 in the purified cells were in the range from 87% to 98%. These purified cells were cultured in serum-free medium with individual colony stimulating factors (CSFs) to investigate whether CD34+ cells from MDS patients have abnormal responses to individual CSFs. Dose response experiments with the purified CD34+ cells and recombinant human macrophage-CSF (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte/macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3) or erythropoietin (rEP) were performed in serum-free fibrin clots in 11 patients. Five patients showed a diminished response to rG-CSF and one patient to rEP. In the remaining six patients the purified CD34+ cells did not respond to a stimulation of any individual CSFs. The results indicate that the progenitor cell growth abnormalities in these disorders involve a defect in the capacity of progenitor cells to respond to stimulation with G-CSF, and present direct evidence for the manner in which myelodysplastic CD34+ cells are impaired.

Effect of stem cell factor (c-kit ligand), granulocyte-macrophage colony stimulating factor and interleukin 3 on hematopoietic progenitors in human long-term bone marrow cultures.

In this paper we attempt to improve upon the methods of hematopoietic stem cell expansion. We evaluate the effects of recombinant human stem cell factor (SCF or c-kit ligand) alone and also in combination with recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 3 (IL-3), on cell proliferation and differentiation in human long term bone marrow cultures (LTBMC). Weekly addition of 5 ng/ml of SCF with 25% serum containing media resulted in increased recovery of total nonadherent cells, granulocyte-macrophage colony forming units (CFU-GM), and burst-forming units erythroid (BFU-E) at week 1, but the number of bone marrow (BM) progenitor cells fell below the level of untreated control cultures at weeks 3 (BFU-E) and 4 (CFU-GM). At week 8, when the cultures were terminated, the CFU-GM recovery was markedly reduced in flasks supplemented with SCF compared with the controls (p < 0.002). Moreover, SCF treatment induced the early disappearance of BFU-E. When LTBMC were supplemented with the combination of SCF plus GM-CSF (100 U/ml) and IL-3 (5 ng/ml), synergistic activity of the CSFs was observed at week 1. The number of BM colony forming cells (CFC) rapidly declined below the level of growth factor-free controls, leading to the early exhaustion of the culture when SCF was combined with GM-CSF. By comparison, GM-CSF and IL-3 alone induced a statistically significant increase above the controls (no growth factor) in the number of nonadherent cell colonies of CFU-GM and BFU-E. Analysis of adherent layer cells from cultures supplemented with SCF showed increased cellularity, no adipogenesis, and early disappearance of myeloid progenitors while the percentage of CFU-GM in S phase, assessed by cytosine arabinoside (Ara-C) suicide assay, was 9.2 +/- 5% SD versus 27.7 +/- 10% SD in control (no growth factor) samples (p < 0.01). SCF increased the number of fibroblast colony forming units (CFU-F) and also showed a synergistic activity (9.6-fold increase) when combined with IL-3. These findings suggest that SCF, GM-CSF and IL-3 exert their activity on different cell populations within the hematopoietic system. Further investigations are needed to optimize the use of SCF in supporting hematopoiesis.

Lymphokine-activated killer cells in autologous bone marrow transplantation. Evidence against inhibition of engraftment in vivo.

Lymphokine activated killer cells have potent antitumor effect both in vitro and in vivo. They have been reported to suppress bone marrow (BM) progenitor cell activity (PCA) in vitro, thus raising concern about the feasibility of their use after autologous bone marrow transplantation. The present study was carried out to evaluate the effect of LAK cells on BM engraftment in a syngeneic BMT setting in mice. LAK cells supplemented with or without exogenous interleukin-2 therapy did not impair the hematopoietic reconstitution or survival of mice undergoing BMT. LAK cells also did not reduce the PCA of the engrafted BM. LAK cell therapy did not cause graft-versus-host disease. Finally, LAK cells supplemented with IL-2 therapy improved the graft-versus-leukemia effect. These findings suggest that LAK cells plus IL-2 therapy after BMT does not impede hematopoiesis and should be evaluated as an adjuvant therapy with the aim of eradication of minimal residual disease after autologous BMT.

Detection of surface antigen expression of pre- and post-cultured normal adult's bone marrow cells by using ABC technique and its significance.

The expression of surface antigens on both normal adult's bone marrow and multipotential progenitor cells (CFU-Mix) was detected by use of ABC technique. The culture system of CFU-Mix was an ideal model for studying the differentiation direction of hematopoietic cells. The monoclonal antibodies (McAb) CD3, CD4, CD8 specific for T-lymphocytic lineage, CD22 for B-lymphocytic lineage and CD11, CD13, CD14 for granulocytomonocytic lineage were all demonstrated in certain proportion in cultured CFU-Mix, which might be associated with the control of hematopoiesis. A decrease in the positive rate of OKT9 and CD34 in cultured cells might be related to the differentiation of hematopoietic cells. SZ-2 and SZ-21 could contribute to the identification of the presence of megakaryocytes in CFU-Mix.

Effects of L‐leucyl‐L‐leucine methyl ester on human marrow and protection of progenitor cells by IL‐1

In murine models, L-leucyl-L-leucine methyl ester (LLME) is effective as an ex vivo purging agent for the prevention of graft-versus-host disease. However, reduction of progenitor cells by LLME raises concerns about the engraftment potential of LLME-treated marrow. Therefore, the effects of LLME on human marrow were investigated. Concentrations of LLME from 0.005 mM to 5 mM were used to examine the effects of LLME on marrow cells. Concentrations of LLME less than or equal to 0.05 mM had no effect on recovery of nucleated marrow cells, mononuclear cells (MNC), myeloid cells or monocytes. At 0.5 mM, nucleated marrow cell recovery was 25%, and MNC recovery was 93%. As determined by cytochemical stains, 0.5 mM LLME eliminated myeloid cells and monocytes. At 0.005 mM, LLME had little effect on marrow progenitor cells; progenitor cell recovery with 0.05 mM LLME was 35% for granulocyte-macrophage colony forming units (CFU-GM) and 23% for erythroid burst forming units (BFU-E) (p less than 0.05 compared to 0.005 mM). At higher LLME concentrations, recoveries of both CFU-GM and BFU-E were less than 1%. To determine if earlier hematopoietic cells remained after treatment with LLME, progenitor cell experiments were repeated with 0.5 mM LLME using recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) or recombinant human interleukin 3 (rhIL-3) plus or minus recombinant human mast cell growth factor (rhMGF) as sources of colony-stimulating activity. In this system, both IL-3 and GM-CSF induced rare progenitor cells (less than 6/5 x 10(4) cells plated).(ABSTRACT TRUNCATED AT 250 WORDS)