Megakaryocyte Growth Research Articles

Increased expansion and differentiation of cord blood products using a two-step expansion culture.

[corrected] The use of allogeneic cord blood (CB) products as a source of cellular support for patients receiving high-dose chemotherapy has been limited primarily to smaller children due to the low numbers of cells in a CB unit. Ex vivo expansion of CB cells has been proposed as a method to increase the number of cells available for transplantation. Following high-dose chemotherapy administration, we transplanted adult patients with CB expanded in static culture for 10 days, in DM containing stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF). Patients achieved neutrophil engraftment in a median of 26 days (range 15 to 45). In an attempt to hasten the time to neutrophil engraftment, we developed a two-step culture system that results in increased expansion of total nucleated cells and further maturation of neutrophil precursors. CD34(+) cells isolated from CB products were cultured for 7 days at 37 degrees C in 100-mL Teflon culture bags containing 50 mL of DM containing SCF, G-CSF, and MGDF (100 ng/mL). The cells were harvested from these bags after 7 days of incubation at 37 degrees C and transferred to 1-L Teflon bags containing 1 L of DM plus SCF, G-CSF, and MGDF. After a second culture period of 7 days, the cells were harvested, washed, and assayed for mature (granulocyte-macrophage colony-forming cells [GM-CFC]) and primitive progenitor cells (high proliferative potential colony-forming cells [HPP-CFC]). The two-step cultures resulted in a median total nucleated cell expansion of 438-fold (range 286 to 952, N = 11); the original one-step cultures resulted in a median expansion of 98-fold (range 59 to 350, N = 5). Equivalent expansion of committed progenitor cells (GM-CFC) and primitive progenitor cells (HPP-CFC) was obtained. CD34(+) cells were expanded a median of 29-fold in the two-step cultures (N = 11). The two-step culture contained more mature neutrophil cells, by morphologic examination, than the one-step cultures, similar to ex vivo expanded peripheral blood progenitor cells (PBPC). The two-step ex vivo expansion conditions described for CB resulted in increased numbers of total nucleated cells, GM-CFC, HPP-CFC, and CD34(+) cells and morphologically resembled ex vivo expanded PBPC, which have been shown to provide more rapid neutrophil engraftment than unexpanded PBPC. We propose that the availability of increased numbers of expanded CB cells may result in more rapid engraftment of neutrophils following infusion to transplant recipients.

Ex vivo expanded unselected peripheral blood: progenitor cells reduce posttransplantation neutropenia, thrombocytopenia, and anemia in patients with breast cancer

AbstractThe safety and efficacy of administering ex vivo expanded peripheral blood progenitor cells (PBPC) to patients with breast cancer who undergo high-dose chemotherapy and PBPC transplantation was investigated. Unselected PBPC were cultured in gas-permeable bags containing 1-L serum-free media, granulocyte colony-stimulating factor, stem cell factor, and pegylated megakaryocyte growth and development factor for 9 days. Cell dose cohorts were assigned to have between 2 and 24 × 109 PBPC cultured at 1, 2, or 3 × 106 cells/mL. Twenty-four patients received high-dose chemotherapy followed by infusion of the cultured PBPC and at least 5 × 106 CD34+ uncultured cryopreserved PBPC per kilogram. No toxicities resulted from infusions of the ex vivo expanded PBPC. The study patients had shorter times to neutrophil (P = .0001) and platelet (P = .01) recovery and fewer red cell transfusions (P = .02) than 48 historical controls who received the same conditioning regimen and posttransplantation care and at least 5 × 106CD34+ PBPC per kilogram. Improvements in all these endpoints were significantly correlated with the expanded cell dose. Nine of 24 (38%) patients recovered neutrophil counts above 500/μL by day 5 or 6 after transplantation, whereas none of the controls had neutrophil recovery before the eighth day. Seven (29%) patients had neutropenia for 3 or fewer days, and 9 (38%) patients did not experience neutropenic fevers or require broad-spectrum antibiotics. Therefore, ex vivo expanded PBPC are capable of ameliorating posttransplantation neutropenia, thrombocytopenia, and anemia in patients receiving high-dose chemotherapy.

Ex vivo expanded unselected peripheral blood: progenitor cells reduce posttransplantation neutropenia, thrombocytopenia, and anemia in patients with breast cancer

The safety and efficacy of administering ex vivo expanded peripheral blood progenitor cells (PBPC) to patients with breast cancer who undergo high-dose chemotherapy and PBPC transplantation was investigated. Unselected PBPC were cultured in gas-permeable bags containing 1-L serum-free media, granulocyte colony-stimulating factor, stem cell factor, and pegylated megakaryocyte growth and development factor for 9 days. Cell dose cohorts were assigned to have between 2 and 24 × 109 PBPC cultured at 1, 2, or 3 × 106 cells/mL. Twenty-four patients received high-dose chemotherapy followed by infusion of the cultured PBPC and at least 5 × 106 CD34+ uncultured cryopreserved PBPC per kilogram. No toxicities resulted from infusions of the ex vivo expanded PBPC. The study patients had shorter times to neutrophil (P = .0001) and platelet (P = .01) recovery and fewer red cell transfusions (P = .02) than 48 historical controls who received the same conditioning regimen and posttransplantation care and at least 5 × 106CD34+ PBPC per kilogram. Improvements in all these endpoints were significantly correlated with the expanded cell dose. Nine of 24 (38%) patients recovered neutrophil counts above 500/μL by day 5 or 6 after transplantation, whereas none of the controls had neutrophil recovery before the eighth day. Seven (29%) patients had neutropenia for 3 or fewer days, and 9 (38%) patients did not experience neutropenic fevers or require broad-spectrum antibiotics. Therefore, ex vivo expanded PBPC are capable of ameliorating posttransplantation neutropenia, thrombocytopenia, and anemia in patients receiving high-dose chemotherapy.

Interferon-α directly represses megakaryopoiesis by inhibiting thrombopoietin-induced signaling through induction of SOCS-1

AbstractInterferon (IFN)-α has proven useful for treating several clinical conditions, including chronic viral hepatitis and chronic myeloproliferative and lymphoproliferative disorders. In addition to its well-known antiviral effects, the cytokine exerts antiproliferative effects on many cell types, helping to explain its therapeutic usefulness in these latter conditions. However, this same property accounts for several undesirable effects, including thrombocytopenia, which can interfere with the successful clinical application of IFN-α. Unfortunately, the mechanisms responsible for the myelosuppressive effects of the cytokine are incompletely understood. The effects of IFN-α on megakaryocyte (MK) development were studied. Using several marrow cell purification techniques and quantitative culture methods, it was found that IFN-α directly inhibits thrombopoietin (TPO)-induced MK growth. Previous studies indicated that Janus kinase (JAK) and its substrates mediate the effects of TPO on cellular proliferation and survival. It was found that IFN-α directly suppresses TPO-induced phosphorylation of the JAK2 substrates c-Mpl and STAT 5 in a TPO-dependent hematopoietic cell line and of Mpl and STAT3 in primary murine MK. Moreover, IFN-α induces SOCS-1 production in these cells, which has been shown to inhibit TPO-induced cell growth. Because SOCS protein expression is induced by many cytokines and has been reported to extinguish signaling from several hematopoietic cytokine receptors, these results identify a molecular mechanism responsible for cytokine receptor cross-talk.

Interferon-α directly represses megakaryopoiesis by inhibiting thrombopoietin-induced signaling through induction of SOCS-1

Interferon (IFN)-α has proven useful for treating several clinical conditions, including chronic viral hepatitis and chronic myeloproliferative and lymphoproliferative disorders. In addition to its well-known antiviral effects, the cytokine exerts antiproliferative effects on many cell types, helping to explain its therapeutic usefulness in these latter conditions. However, this same property accounts for several undesirable effects, including thrombocytopenia, which can interfere with the successful clinical application of IFN-α. Unfortunately, the mechanisms responsible for the myelosuppressive effects of the cytokine are incompletely understood. The effects of IFN-α on megakaryocyte (MK) development were studied. Using several marrow cell purification techniques and quantitative culture methods, it was found that IFN-α directly inhibits thrombopoietin (TPO)-induced MK growth. Previous studies indicated that Janus kinase (JAK) and its substrates mediate the effects of TPO on cellular proliferation and survival. It was found that IFN-α directly suppresses TPO-induced phosphorylation of the JAK2 substrates c-Mpl and STAT 5 in a TPO-dependent hematopoietic cell line and of Mpl and STAT3 in primary murine MK. Moreover, IFN-α induces SOCS-1 production in these cells, which has been shown to inhibit TPO-induced cell growth. Because SOCS protein expression is induced by many cytokines and has been reported to extinguish signaling from several hematopoietic cytokine receptors, these results identify a molecular mechanism responsible for cytokine receptor cross-talk.

Highly efficient gene transfer into preterm CD34+ hematopoietic progenitor cells

Objective: Retrovirus-mediated gene transfer has been shown to transduce CD34+ cells from term gestation umbilical cord blood with relatively high efficiency. The purpose of this study was to compare the efficiencies of retrovirus-mediated gene transfer into early (23-28 weeks’ gestation) and term (37-41 weeks’ gestation) umbilical cord blood CD34+ hematopoietic progenitor cells. Study Design: CD34+ cells were purified from cyropreserved early (23-28 weeks’ gestation) and term (37-40 weeks’ gestation) umbilical cord blood specimens with fluorescence-activated cell sorting. The CD34+ cells were then transduced in virus-containing medium (gibbon ape leukemia virus pseudotype vector LAPSN [PG13]) in wells coated with the recombinant human fibronectin fragment CH-296 and in the presence of multiple hematopoietic growth factors (interleukin 6, stem cell factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and megakaryocyte growth and development factor) and protamine sulfate. The LAPSN (PG13) virus–containing medium was changed every 12 hours for 48 hours, after which time colony-forming cells were assayed in soft agar. The gibbon ape leukemia virus pseudotype vector LAPSN (PG13) contains the human placental alkaline phosphatase and neomycin phosphotransferase (neo ) genes. The efficiency of gene transfer was assessed by histochemical staining of colony-forming cells in agar for expression of heat-stable alkaline phosphatase. Results: Gene transfers, as assessed by alkaline phosphatase staining of colony-forming cells (granulocyte-macrophage colony-forming units and erythroid burst-forming units), were similar for CD34+ hematopoietic progenitor cells from early (58.4% ± 11.8%) and term (63.2% ± 12.5%) gestation fetal umbilical cord blood. Conclusion: CD34+ hematopoietic progenitor cells from midgestation fetal blood can be transduced with high efficiency using techniques optimized for postnatal samples with a gibbon ape leukemia virus pseudotype vector. The early fetus may be a preferable target for gene therapy because of the higher number of circulating CD34+ and CD38– cells relative to term cord blood, their greater proliferative capacity, and the rapid expansion of the fetal hematopoietic system that occurs from the second trimester to delivery. Because in vitro studies of gene transfer into hematopoietic progenitor cells and long-term culture-initiation cells have not been predictive of the efficiency of gene transfer into marrow-repopulating cells in vivo, studies that examine clinically applicable approaches to in utero gene therapy in appropriate animal models are still needed. (Am J Obstet Gynecol 2000;183:732-7.)

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

AbstractAlthough long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

Enhancement of Platelet Recovery After Myelosuppressive Chemotherapy by Recombinant Human Megakaryocyte Growth and Development Factor in Patients With Advanced Cancer

To explore the influence of dose and schedule on the ability of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) to abrogate thrombocytopenia after multiple cycles of chemotherapy and to mobilize peripheral-blood progenitor cells (PBPC). In this open-label study, 68 patients with advanced cancer were randomized to receive PEG-rHuMGDF subcutaneously at different doses and durations before administration of carboplatin 600 mg/m(2), cyclophosphamide 1,200 mg/m(2), and filgrastim 5 microgram/kg/d. PEG-rHuMGDF was not given after the first cycle of chemotherapy but was given after the second and subsequent cycles. Chemotherapy was given every 28 days for up to six cycles. In patients who received the same dose of chemotherapy for at least two cycles, the platelet nadir was significantly higher (47.5 x 10(9)/L v 35.5 x 10(9)/L; P =.003) and duration of grade 3 or 4 thrombocytopenia significantly shorter (0 v 3 days; P =.004) when PEG-rHuMGDF was administered after chemotherapy. There was no evidence of an effect of PEG-rHuMGDF when it was given before chemotherapy. Platelet recovery after the first cycle of chemotherapy was no different for different PEG-rHuMGDF regimens, and there was no difference between patients treated with PEG-rHuMGDF and historical controls treated with identical chemotherapy. There was a modest dose-related increase in progenitor cell levels after administration of PEG-rHuMGDF alone. Peak levels of PBPC occurred later in cycle 2 than in cycle 1 but were not different in magnitude. PEG-rHuMGDF abrogated severe thrombocytopenia after dose-intensive chemotherapy. However, it had only a modest effect on progenitor cell levels and did not enhance progenitor cell mobilization after chemotherapy and filgrastim.

Thrombopoietin and the TPO receptorduring platelet storage.

For most cells, the addition of a specific growth factor has improved cellular viability by preventing programmed cell death (apoptosis). To determine whether the platelet-specific hematopoietic growth factor thrombopoietin (TPO) might improve platelet viability, endogenous TPO and the platelet TPO receptor were analyzed during storage, and the effect of recombinant TPO on platelet viability was assessed. During platelet storage, TPO stability was assessed by SDS-PAGE, TPO receptor function was measured, and the platelet TPO receptor was characterized by a (125)I-rHuTPO competitive-binding assay. A recombinant TPO, pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), was added to platelet concentrates during storage, and its effect on pH, LDH, and metabolic activity was determined. During storage, the molecular weight and concentration of endogenous TPO (125 +/- 19 pg/mL) and exogenous TPO (5720 +/- 140 pg/mL) were constant for 12 days; the number (33 +/- 4), binding affinity (149 +/- 33 pM), and function of the platelet TPO receptors were constant for 7 days. Metabolic activity measured with the MTT and MTS assays closely correlated with changes in the pH and LDH. The addition of PEG-rHuMGDF did not alter the pH, LDH, or metabolic activity of platelets during storage, but it did increase by 65 percent the uptake of (35)S-methionine into platelets. Finally, platelet concentrates obtained from donors treated with PEG-rHuMGDF retained normal metabolic activity for 12 days, as compared with 5 to 6 days for normal platelet concentrates. TPO and its platelet receptor are present in normal amounts and have normal function during platelet storage. The addition of recombinant TPO increased platelet methionine transport but did not alter platelet viability during storage. Other means to prevent apoptosis during platelet storage should be considered, and the measurement of platelet metabolic activity by MTT and MTS assays may assist this effort.

Cocultivation of umbilical cord blood cells with endothelial cells leads to extensive amplification of competent CD34 +CD38 − cells

Objective In this report, methods to expand the number of human cord blood hematopoietic stem cells were explored. Materials and Methods CD34 + cord blood cells were expanded in the presence of various cytokine combinations in either a stroma-free cell culture system or a preformed porcine microvascular endothelial cell layer. After 7 to 21 days, stem cell number and function were monitored. In addition, the replicative history of stem cells was tracked using the fluorescent dye, PKH26. Results With the addition of various cytokine combinations, total cellular expansion was equivalent for both culture systems, although the endothelial cell–based system contained statistically greater numbers of CD34 + cells. By day 21, the endothelial-based system receiving the FLT3L, SCF, IL-6, and GM-CSF cytokine combination contained five-fold greater numbers of CD34 + than the stroma cell–free culture cell system. Endothelial-based cultures receiving these four cytokines plus megakaryocyte growth and development factor produced a 640-fold expansion of CD34 + CD38 − cells as compared to a four-fold expansion in the stroma-free system. The number of progenitor cells generated was similar with both systems, yet the greatest degree of expansion of cobblestone area–forming cells was observed in the endothelial based cultures (11-fold vs four-fold). Virtually all CD34 + and CD34 + CD38 + cells expanded in the presence of endothelial cells had undergone self replication by day 10, yet stromal cell–free cultures contained a significant number (4.8%) of quiescent cells. Identical numbers of re-isolated cord blood CD34 + cells expanded in both systems exhibited a similar ability to engraft and generate cells belonging to multiple hematopoietic lineages in human fetal bones implanted in immunodeficient mice. Conclusions These results suggest that the use of preformed endothelial cell monolayers might permit the ex vivo generation of sufficient numbers of cord stem cells to serve as successful grafts for adult transplant recipients.

The antiapoptotic function of megakaryocyte growth and development factor (Peg-rmMGDF) Is disrupted in STAT5ab−/− and JAK2+/− mice

PEG-rmMGDF (60 μg/kg) given to mice immediately after lethal myelosuppression (Carboplatin 80 mg/kg + 750R Cs-137), prevents death and promotes multilineage hematopoietic reconstitution (Pestina et al., Exp Hematol 27, Suppl 1: 100, 1999). Our data from study of p53-deficient mice suggests that this myeloprotection results in part by preventing p53-dependent apoptosis of c-Mpl-expressing multipotential hematopoietic cells. To define molecules involved in the antiapoptotic signaling pathways induced by c-Mpl ligation, we performed in vivo studies using mice with specific gene deletions. Jak-2 tyrosine kinase is rapidly activated as a result of c-Mpl ligation, but its role in antiapoptotic signaling has not been examined in vivo. Because Jak2−/− is an embryonic lethal due to failed development of hematopoiesis, we examined whether adult Jak2+/− mice were protected from lethal myelosuppression by PEG-rmMGDF. The survival rate of Jak2+/− mice (68%) was lower than that of wild-type (WT) mice (91%), indicating that Jak2 activation plays a role in the myeloprotective signaling pathway in vivo. We next investigated the importance of the Jak2 substrates Stat5a and b in this pathway using mice with combined deficiency of these Stat proteins. Intact Stat5ab−/− mice had normal steady-state platelet counts and similar elevations in platelet numbers in response to PEG-rmMGDF to those of WT mice, suggesting that neither Stat5 protein is required during megakaryocytopoieses. However, Stat5−/− mice exposed to the myelosuppressive regimen were severely compromised in their ability to be protected by PEG-rmMGDF (19% survival vs. 100% for Stat5ab+/− and 90% for WT mice). These findings provide in vivo evidence that Jak2 and Stat5 play important roles in the myeloprotective signaling pathways initiated by c-Mpl ligation.

Megakaryocyte-targeted synthesis of the integrin β3-subunit results in the phenotypic correction of Glanzmann thrombasthenia

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, IIbβ3. As a result, IIbβ3 cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, −889PlA2β3, was transduced into peripheral blood CD34+ cells from 2 patients with thrombasthenia with defects in the β3 gene. The human IIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type β3 subunit. Proviral DNA and IIbβ3 biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34+ cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed IIbβ3 on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the IIbβ3 complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34+ cells as targets for IIb promoter-driven MuLV vectors for gene therapy of platelet disorders.

Nitric oxide induces apoptosis in megakaryocytic cell lines

AbstractCytokines that stimulate inducible nitric oxide (NO) synthase can suppress the growth and differentiation of normal human bone marrow cells, including megakaryocytes. Since NO promotes apoptosis in other cell systems, we chose to study the determinants of apoptosis in megakaryocytic cells. We show that both exogenous and endogenous sources of NO can induce apoptosis in megakaryocytoid cell lines. The megakaryocyte growth factor thrombopoietin suppresses NO-induced apoptosis, whereas treatment with peroxynitrite, a cytotoxic product formed when NO reacts with superoxide, promotes apoptosis. Superoxide inhibitors suppress NO-induced apoptosis, and pretreatment with megakaryocyte growth and maturation factors attenuates NO-induced apoptosis. These data show that NO modulates megakaryocyte apoptosis and suggest that this process may occur in the cytokine-rich marrow milieu to regulate megakaryocyte turnover.

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.

Nitric oxide induces apoptosis in megakaryocytic cell lines

Cytokines that stimulate inducible nitric oxide (NO) synthase can suppress the growth and differentiation of normal human bone marrow cells, including megakaryocytes. Since NO promotes apoptosis in other cell systems, we chose to study the determinants of apoptosis in megakaryocytic cells. We show that both exogenous and endogenous sources of NO can induce apoptosis in megakaryocytoid cell lines. The megakaryocyte growth factor thrombopoietin suppresses NO-induced apoptosis, whereas treatment with peroxynitrite, a cytotoxic product formed when NO reacts with superoxide, promotes apoptosis. Superoxide inhibitors suppress NO-induced apoptosis, and pretreatment with megakaryocyte growth and maturation factors attenuates NO-induced apoptosis. These data show that NO modulates megakaryocyte apoptosis and suggest that this process may occur in the cytokine-rich marrow milieu to regulate megakaryocyte turnover.

Effects of megakaryocyte growth and development factor on platelet production, platelet life span, and platelet function in healthy human volunteers

The effects of thrombopoietic stimulation on megakaryocytopoiesis, platelet production, and platelet viability and function were examined in normal volunteers randomized to receive single bolus subcutaneous injections of 3 μg/kg pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo in a 3:1 ratio. PEG-rHuMGDF transiently doubled circulating platelet counts, from 237 ± 41 × 103/μL to 522 ± 90 × 103/μL (P< .0001), peaking on day 12. Baseline and day-12 samples showed no differences in responsiveness of platelets to adenosine diphosphate or thrombin receptor agonist peptide (P > .4 in all cases); expression of platelet ligand-induced binding sites or annexin V binding sites (P > .6 in both cases); or density of platelet TPO-receptors (P > .5). Platelet counts normalized by day 28. The life span of autologous 111In-labeled platelets increased from 205 ± 18 hours (baseline) to 226 ± 22 hours (P < .01) on day 8. Platelet life span decreased from 226 ± 22 hours (day 8) to 178 ± 53 hours (P < .05) on day 18. The theoretical basis for senescent changes in mean platelet life span was illustrated by biomathematical modeling. Platelet turnover increased from 43.9 ± 11.9 × 103 platelets/μL/d (baseline) to 101 ± 27.6 × 103 platelets/μL/d (P = .0009), and marrow megakaryocyte mass expanded from 37.4 ± 18.5 fL/kg to 62 ± 17 × 1010 fL/kg (P = .015). Although PEG-rHuMGDF initially increased megakaryocyte volume and ploidy, subsequently ploidy showed a transient reciprocal decrease when the platelet counts exceeded placebo values. In healthy human volunteers PEG-rHuMGDF transiently increases megakaryocytopoiesis 2-fold. Additionally, peripheral platelets expand correspondingly and exhibit normal function and viability during the ensuing 10 days. The induced perturbation in steady state thrombopoiesis resolves by 4 weeks.

Effects of megakaryocyte growth and development factor on platelet production, platelet life span, and platelet function in healthy human volunteers

AbstractThe effects of thrombopoietic stimulation on megakaryocytopoiesis, platelet production, and platelet viability and function were examined in normal volunteers randomized to receive single bolus subcutaneous injections of 3 μg/kg pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo in a 3:1 ratio. PEG-rHuMGDF transiently doubled circulating platelet counts, from 237 ± 41 × 103/μL to 522 ± 90 × 103/μL (P< .0001), peaking on day 12. Baseline and day-12 samples showed no differences in responsiveness of platelets to adenosine diphosphate or thrombin receptor agonist peptide (P > .4 in all cases); expression of platelet ligand-induced binding sites or annexin V binding sites (P > .6 in both cases); or density of platelet TPO-receptors (P > .5). Platelet counts normalized by day 28. The life span of autologous 111In-labeled platelets increased from 205 ± 18 hours (baseline) to 226 ± 22 hours (P < .01) on day 8. Platelet life span decreased from 226 ± 22 hours (day 8) to 178 ± 53 hours (P < .05) on day 18. The theoretical basis for senescent changes in mean platelet life span was illustrated by biomathematical modeling. Platelet turnover increased from 43.9 ± 11.9 × 103 platelets/μL/d (baseline) to 101 ± 27.6 × 103 platelets/μL/d (P = .0009), and marrow megakaryocyte mass expanded from 37.4 ± 18.5 fL/kg to 62 ± 17 × 1010 fL/kg (P = .015). Although PEG-rHuMGDF initially increased megakaryocyte volume and ploidy, subsequently ploidy showed a transient reciprocal decrease when the platelet counts exceeded placebo values. In healthy human volunteers PEG-rHuMGDF transiently increases megakaryocytopoiesis 2-fold. Additionally, peripheral platelets expand correspondingly and exhibit normal function and viability during the ensuing 10 days. The induced perturbation in steady state thrombopoiesis resolves by 4 weeks.

A double-blind, placebo-controlled trial of pegylated recombinant human megakaryocyte growth and development factor as an adjunct to induction and consolidation therapy for patients with acute myeloid leukemia

AbstractNewly diagnosed patients with acute myeloid leukemia (AML) were randomized to receive either 2.5 or 5 μg/kg/day of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or a placebo administered subcutaneously after completion of chemotherapy. The study evaluated the toxicity of PEG-rHuMGDF and any effect on the duration of thrombocytopenia. Each of 35 patients under 60 years of age received the following therapy: 45 mg/m2 daunorubicin on days 1-3, 100 mg/m2cytarabine (ARA-C) for 7 days, and 2 gm/m2 high-dose ARA-C (HIDAC) for 6 doses on days 8-10. The 22 patients 60 years or older received standard daunorubicin and ARA-C without HIDAC. PEG-rHuMGDF was well tolerated, and no specific toxicities could be attributed to its use. There was no difference in the time to achieve a platelet count of at least 20 × 109/L among the 3 groups (median 28-30 days for patients less than 60 years old and 21-23 days for patients 60 years or older). Patients receiving PEG-rHuMGDF achieved higher platelet counts after remission. However there was no significant difference in the number of days on which platelet transfusions were administered among the 3 groups. The complete remission rate was 71% for patients less than 60 years and 64% for those 60 years or older, with no significant difference among the 3 groups. Postremission consolidation chemotherapy with either placebo or PEG-rHuMGDF was given to 28 patients beginning the day after completion of chemotherapy. There was no apparent difference in the time that was necessary to reach a platelet count of at least 20 or 50 × 109/L or more platelets or in the number of platelet transfusions received. In summary, PEG-rHuMGDF was well tolerated by patients receiving induction and consolidation therapy for AML; however, there was no effect on the duration of severe thrombocytopenia or the platelet transfusion requirement.

A double-blind, placebo-controlled trial of pegylated recombinant human megakaryocyte growth and development factor as an adjunct to induction and consolidation therapy for patients with acute myeloid leukemia

Newly diagnosed patients with acute myeloid leukemia (AML) were randomized to receive either 2.5 or 5 μg/kg/day of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or a placebo administered subcutaneously after completion of chemotherapy. The study evaluated the toxicity of PEG-rHuMGDF and any effect on the duration of thrombocytopenia. Each of 35 patients under 60 years of age received the following therapy: 45 mg/m2 daunorubicin on days 1-3, 100 mg/m2cytarabine (ARA-C) for 7 days, and 2 gm/m2 high-dose ARA-C (HIDAC) for 6 doses on days 8-10. The 22 patients 60 years or older received standard daunorubicin and ARA-C without HIDAC. PEG-rHuMGDF was well tolerated, and no specific toxicities could be attributed to its use. There was no difference in the time to achieve a platelet count of at least 20 × 109/L among the 3 groups (median 28-30 days for patients less than 60 years old and 21-23 days for patients 60 years or older). Patients receiving PEG-rHuMGDF achieved higher platelet counts after remission. However there was no significant difference in the number of days on which platelet transfusions were administered among the 3 groups. The complete remission rate was 71% for patients less than 60 years and 64% for those 60 years or older, with no significant difference among the 3 groups. Postremission consolidation chemotherapy with either placebo or PEG-rHuMGDF was given to 28 patients beginning the day after completion of chemotherapy. There was no apparent difference in the time that was necessary to reach a platelet count of at least 20 or 50 × 109/L or more platelets or in the number of platelet transfusions received. In summary, PEG-rHuMGDF was well tolerated by patients receiving induction and consolidation therapy for AML; however, there was no effect on the duration of severe thrombocytopenia or the platelet transfusion requirement.