In vitro large scale production of megakaryocytes to functional platelets from human hematopoietic stem cells.

Megakaryocytopoiesis results in the formation of platelets, which are essential for hemostasis. Decreased production or increased destruction of platelets can cause thrombocytopenia, in which platelet transfusion is the mode of treatment. The present study is aimed in generation of megakaryocytes (MKs) and platelet from human hematopoietic stem cells (HSCs). The purity of HSCs was assessed through Flow cytometry and immunocytochemistry (ICC) studies. These pure HSCs were induced with thrombopoietin (TPO), similarly with Andrographis paniculata extract (APE) for 21 days to generate MKs. The APE is mainly composed of andrographolide which stimulates TPO from the liver, and this binds to CD110 present on the surface of HSCs and triggers the proliferation of HSCs and initiate higher MKs population subsequently, a large number of platelets. The results of the present study showed increased proliferation of HSCs grown in the presence of APE and revealed a high population of CD41a and CD42b positive MKs as enumerated by Flow cytometry compared with TPO induced MKs. These results also concurred with qRT-PCR and western blot analysis. The scanning electron microscopy (SEM) revealed the morphology of differentiated MKs and platelets were similar to human blood platelets. The differentiated MKs in APE exhibited polyploidy up to 32 N while TPO induced MKs showed polyploidy of 8 N, these results corroborated with colony forming unit assay. On thrombin stimulation, high expression of P-selectin (CD62p) and fibrinogen binding were detected in APE induced platelets. Autologous transplantation of platelets generated from APE may be a useful option in thrombocytopenia condition.

[Anti-apoptotic effect of Astragalus Polysaccharide on myeloid cells].

This study was aimed to assess the effect of Astragalus Polysaccharide (ASPS) on in-vitro hematopoiesis. CFU-GM assays were used to determine the effect of ASPS and thrombopoietin (TPO) on granulocytic-monocyte progenitor cells. The CFU assays were also used to investigate the effect of ASPS on the proliferation of HL-60 cells.HL-60 cells were cultured with serum-free RPMI 1640 medium and treated with or without of different concentrations of ASPS. After 72 h incubation, the number of cells were counted.In addition, the caspase-3 and JC-1 expression was determined by flow cytometry with Annexin V/PI double staining. The results showed that ASPS (100, 200 µg/ml) and TPO (100 ng/ml) significantly promoted CFU-GM formation in vitro. Various concentrations of ASPS and TPO also promoted the colony formation of HL-60 cells, the largest effect of ASPS was observed at a concentration of 100 µg/ml. There were no synergistic effects between TPO and ASPS on cellular proliferation. The results also showed that ASPS significantly protected HL-60 cells from apoptosis in condition of serum-free medium culture, suppressed caspase 3 activation, and reduced the cell apoptosis. It is concluded that ASPS can significantly promote the formation of bone marrow CFU-GM and the proliferation of HL-60 cells, the optimal concentration of ASPS is at 100 µg/ml. In the absence of serum inducing apoptosis, ASPS also significantly reduced the apoptosis of HL-60 cells via suppressing the activation of caspase-3.

A novel and simple hollow-fiber assay for in vivo evaluation of nonpeptidyl thrombopoietin receptor agonists.

Preclinical in vivo assessment of the pharmacologic activity of nonpeptidyl thrombopoietin receptor (TPOR) agonists is very difficult because of the high species specificity of such agonists. In this study, we have developed a novel and simple in vivo hollow-fiber assay to preclinically evaluate TPOR agonists. The 32D-mpl cell line was generated by stable transfection of human TPOR into 32D lymphoblast cells and shown to be a specific model for nonpeptide TPOR agonists in vitro. Stably transfected 32D-mpl cells were then sealed in hollow fibers and implanted into nude mice. Cells in hollow fibers specifically responded to TPOR agonists, including thrombopoietin and eltrombopag, a nonpeptide small-molecule TPOR agonist, but not to granulocyte colony-stimulating factor or erythropoietin. Oral administration of eltrombopag stimulated 32D-mpl cell proliferation, prevented 32D-mpl cell apoptosis, and stimulated the phosphorylation of cellular signaling transducers and activators of transcription in a TPOR- and dose-dependent manner. These results indicate that the hollow-fiber assay is a specific and efficient model for rapidly evaluating the in vivo activity of small-molecule TPOR agonists.

Ex vivo expansion of human hematopoietic stem cells by a small-molecule agonist of c-MPL.

OBJECTIVE: The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs. MATERIALS AND METHODS: hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays. RESULTS: During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes. CONCLUSION: This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.

CFU-megakaryocytic progenitors expanded ex vivo from cord blood maintain their in vitro homing potential and express matrix metalloproteinases.

BACKGROUND: In patients transplanted with cord blood (CB), prolonged thrombocytopenia is a major complication. However, this could be alleviated by supplementing the CB graft with ex vivo-expanded megakaryocytic progenitors (CFU-Meg), provided that the homing properties of these cells are not affected negatively by expansion. METHODS AND RESULTS: We assessed the in vitro homing potential of CFU-Meg progenitors expanded from CB and showed that the combination of thrombopoietin (TPO) with interleukin-3 (IL-3) used for expansion not only results in optimal proliferation of CFU-Meg but also protects these cells from apoptosis. Moreover, we found that ex vivo-expanded CFU-Meg maintained expression of the CXCR4 receptor throughout a 9-day culture and were chemoattracted towards a stromal cell-derived factor-1 (SDF-1) gradient. They also expressed matrix metalloproteinase-9 (MMP-9) and membrane-type (MT) 1-MMP, and transmigrated across the reconstituted basement membrane Matrigel. Finally, we observed that SDF-1 up-regulated the expression of both MMP-9 and MT1-MMP in CB CD34(+) cells and ex vivo-expanded CFU-Meg. DISCUSSION: We suggest that CB-expanded CFU-Meg, in particular those from day 3 of expansion, when their proliferation and in vitro homing potential are maximal, could be employed to supplement CB grafts and speed up platelet recovery in transplant recipients.

Thrombopoietin protects against in vitro and in vivo cardiotoxicity induced by doxorubicin.

BACKGROUND: Doxorubicin (DOX) is an important antineoplastic agent. However, the associated cardiotoxicity, possibly mediated by the production of reactive oxygen species, has remained a significant and dose-limiting clinical problem. Our hypothesis is that the hematopoietic/megakaryocytopoietic growth factor thrombopoietin (TPO) protects against DOX-induced cardiotoxicity and might involve antiapoptotic mechanism exerted on cardiomyocytes. METHODS AND RESULTS: In vitro investigations on H9C2 cell line and spontaneously beating cells of primary, neonatal rat ventricle, as well as an in vivo study in a mouse model of DOX-induced acute cardiomyopathy, were performed. Our results showed that pretreatment with TPO significantly increased viability of DOX-injured H9C2 cells and beating rates of neonatal myocytes, with effects similar to those of dexrazoxane, a clinically approved cardiac protective agent. TPO ameliorated DOX-induced apoptosis of H9C2 cells as demonstrated by assays of annexin V, active caspase-3, and mitochondrial membrane potential. In the mouse model, administration of TPO (12.5 microg/kg IP for 3 alternate days) significantly reduced DOX-induced (20 mg/kg) cardiotoxicity, including low blood cell count, cardiomyocyte lesions (apoptosis, vacuolization, and myofibrillar loss), and animal mortality. Using Doppler echocardiography, we observed increased heart rate, fractional shortening, and cardiac output in animals pretreated with TPO compared with those receiving DOX alone. CONCLUSIONS: These data have provided the first evidence that TPO is a protective agent against DOX-induced cardiac injury. We propose to further explore an integrated program, incorporating TPO with other protocols, for treatment of DOX-induced cardiotoxicity and other forms of cardiomyopathy.

In vitro expansion of long-term repopulating hematopoietic stem cells in the presence of immobilized Jagged-1 and early acting cytokines.

There is an increasing body of evidence that suggests that genes involved in cell fate decisions and pattern formation during development also play a key role in the continuous cell fate decisions made by adult tissue stem cells. Here we show that prolonged in vitro culture (14 days) of murine bone marrow lineage negative cells in medium supplemented with three early acting cytokines (stem cell factor, Flk-2/Flt-3 ligand, thrombopoietin) and with immobilized Notch ligand, Jagged-1, resulted in robust expansion of serially transplantable hematopoietic stem cells with long-term repopulating ability. We found that the absolute number of marrow cells was increased approximately 8 to 14-fold in all cultures containing recombinant growth factors. However, the frequency of high quality stem cells was markedly reduced at the same time, except in cultures containing growth factors and Jagged-1-coated Sepharose-4B beads. The absolute number of hematopoietic cells with long-term repopulating ability was increased approximately 10 to 20-fold in the presence of multivalent Notch ligand. These results support a role for combinatorial effects by Notch and cytokine-induced signaling pathways in regulating hematopoietic stem cell fate and to a potential role for Notch ligand in increasing cell numbers in clinical stem cell transplantation.

Increased plasma levels of stromal-derived factor-1 (SDF-1/CXCL12) enhance human thrombopoiesis and mobilize human colony-forming cells (CFC) in NOD/SCID mice.

OBJECTIVE: Stromal-derived factor-1 (SDF-1/CXCL12) is chemotactic for lympho/hematopoietic stem cells. We have previously shown that increasing peripheral blood (PB) levels of SDF-1 with adenovectors expressing human SDF-1 complementary DNA (ad-SDF-1) leads to hematopoietic stem cell mobilization as well as migration of megakaryocytes and thrombocytosis in mice. Herein, we studied the in vivo effects of ad-SDF-1 and of an analogue peptide of SDF-1 (CTCE-0214) on human hematopoiesis in a xenotransplant model. MATERIALS AND METHODS: Sublethally irradiated (300 cGY) NOD/SCID mice transplanted with human cord blood mononuclear cells (CB MNC) were injected with ad-SDF-1 (10(9) plaque forming units, i.v., x 1) or CTCE-0214 (10 mg/kg/dose, i.v. q 24 hours x 7). Effects on megakaryocytopoiesis (CD41+ cells and platelets) as well as stem cell mobilization were monitored. RESULTS: CB MNC in NOD/SCID mice are able to differentiate into CD41+ cells and platelets, peaking at week 9 at a mean of 3.7 x 10(3)/microL. i.v. injection of ad-SDF-1 increased human CD41+ cells by day 4 in PB and was followed by an increase in human platelet production by day 5, with return to baseline by day 30. Human colony-forming cells (CFC) were mobilized from bone marrow to spleen (by day 6-13) and to PB (by day 13). Human CD34+ and CD33+ cells were mobilized by this treatment as well. A novel SDF-1 peptide agonist (CTCE-0214) also mobilized human CFC and enhanced human thrombopoiesis. CONCLUSION: SDF-1 and its analogue may be of clinical value in stimulating platelet recovery after chemo/radiation treatment as well as in stem cell mobilization.

Hepatocyte growth factor exerts a proliferative effect on oval cells through the PI3K/AKT signaling pathway.

Hepatocyte growth factor (HGF) is a potent mitogen for a variety of cells including hepatocytes. While rat oval cells are supposed to be one of hepatic stem cells, biological effects of HGF on oval cells and their relevant signal transduction pathways remain to be determined. We sought to investigate them on OC/CDE22 rat oval cells, which are established from the liver of rats fed a choline-deficient/DL-ethionine-supplemented diet. The oval cells were cultured on fibronectin-coated dishes and stimulated with recombinant HGF, transforming growth factor-alpha (TGF-alpha), and thrombopoietin (TPO) under the serum-free medium condition. HGF treatment enhanced [3H]thymidine incorporation into oval cells in a dose-dependent manner. On the contrary, treatment with TGF-alpha or TPO had no significant effects on [3H]thymidine incorporation into the oval cells. c-Met protein was phosphorylated at the tyrosine residues after the HGF treatment. AKT, extracellular signal-regulated kinase 1/2 (ERK1/2), and p70(s6k) were simultaneously activated after the HGF stimulation, peaking at 30min after the treatment. The activation of AKT, p70(s6k), and ERK1/2 induced by HGF was abolished by pre-treatment with LY294002, a phosphoinositide 3-OH kinase (PI3K) inhibitor, and U0126, a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, respectively. When the cells were pre-treated with LY294002 prior to the HGF stimulation, the proliferative action of HGF was completely abrogated, implying that the PI3K/AKT signaling pathway is responsible for the biological effect of HGF. These in vitro data indicate that HGF exerts a proliferative action on hepatic oval cells via activation of the PI3K/AKT signaling pathway.

Effect of c-mpl ligands after total body irradiation (TBI) with and without allogeneic hematopoietic stem cell transplantation: low-dose TBI does not prevent sensitization.

This study investigates the potential role of the recombinant c-mpl ligands (recombinant human thrombopoietin [rhTPO] and pegylated recombinant human megakaryocyte growth and development factor [PEG-rhMGDF]) on the recovery of platelet counts after TBI with and without allogeneic hematopoietic stem cell transplantation (HSCT) in an established canine model. Initially, 3 cohorts, each with 2 nonirradiated dogs, received increasing doses of rhTPO (5 microg/kg per day; 10 microg/kg per day; 20 microg/kg per day) for 7 days to determine the optimal dose. The dose of 10 microg/kg per day of rhTPO was selected for subsequent studies. Ten dogs then received either rhTPO or placebo for 28 days after 200 cGy TBI without HSCT. The rhTPO group had fewer days with platelet counts <20,000/microL (9.8 days versus 17.8 days, P < .05) and significantly increased granulocyte counts (n = 5) compared to the controls (n = 5). RhTPO-specific antibodies developed in 2 dogs, which caused a significant but transient decrease of the platelet counts. Retreatment of these sensitized dogs with rhTPO resulted in profound transient decreases in platelet counts. In the next study, 20 dogs received either PEG-rhMGDF or placebo for 21 days after 920 cGy TBI and allogeneic HSCT. The median time to platelet recovery (>20,000/microL) for the PEG-rhMGDF group (n = 10) was 14.0 days compared to 15.5 days for the control group (n = 10; log rank, P = .35). There were no significant differences in the total time to platelet counts <20,000/microL or in the time to recover neutrophil counts >500/microL. The effects of rhTPO on recovery of platelet and granulocyte counts after sublethal TBI were modest, and no effects of PEG-rhMGDF were observed on hematopoietic recovery after high-dose TBI and allogeneic HSCT. The significant effect that rhTPO-specific antibodies had on the platelet counts may limit the clinical role of recombinant c-mpl ligands unless sensitization can be prevented.

Hematopoietic growth factor mimetics.

Hematopoietic growth factors are glycoproteins of 15-70 kDa. Although much clinical success has been obtained using recombinant proteins produced in mammalian cell lines and in microbial fermentation processes, the full-length polypeptides necessarily are expensive to produce, require parenteral administration, and in some cases have provoked detrimental immune responses. With the availability of high throughput biological function and receptor binding assays it has become possible to screen millions, if not billions, of randomly produced organic compounds and relatively short peptides to identify lead compounds for the development of small molecular mimetics of hematopoietic growth factors. Herein the strategies used to screen libraries of small molecules and peptides and the successes in finding mimetics and antagonists for/to erythropoietin, granulocyte colony-stimulating factor, and thrombopoietin are reviewed. Finally, the structural study of mimetic-receptor complexes has provided us with many molecular details of growth factor-induced receptor activation and is likely to yield new insights into the molecular basis of hematopoietic signal transduction.

Single administration of thrombopoietin to lethally irradiated mice prevents infectious and thrombotic events leading to mortality.

A sufficiently high dose of thrombopoietin to overcome initial c-mpl-mediated clearance stimulates hematopoietic reconstitution following myelosuppressive treatment. We studied the efficacy of thrombopoietin on survival after supralethal total body irradiation (9 Gy) of C57BL6/J mice and the occurrence of infectious and thrombotic complications in comparison with a bone marrow graft or prophylactic antibiotic treatment. Administration of 0.3 microg thrombopoietin, 2 hours after irradiation, protected 62% of the mice as opposed to no survival in placebo controls. A graft with a supraoptimal number of syngeneic bone marrow cells (10(6) cells) fully prevented mortality, whereas antibiotic treatment was ineffective. Blood cell recovery was observed in the thrombopoietin-treated mice but not in the placebo or antibiotic-treated group. Bone marrow and spleen cellularity as well as colony-forming unit granulocyte-macrophage and burst-forming unit erythroid were considerably increased in thrombopoietin-treated mice relative to controls. Histologic examination at day 11 revealed numerous petechiae and vascular obstructions within the brain microvasculature of placebo-treated mice, which was correlated with hypercoagulation and hypofibrinolysis. Thrombopoietin treatment prevented coagulation/fibrinolysis disorder and vascular thrombosis. High fibrinogen levels were related to bacterial infections in 67% of placebo-treated mice and predicted mortality, whereas the majority of the thrombopoietin-treated mice did not show high fibrinogen levels and endotoxin was not detectable in plasma. We conclude that thrombopoietin administration prevents mortality in mice subjected to 9-Gy total body irradiation both by interfering in the cascade leading to thrombotic complications and by amelioration of neutrophil and platelet recovery and thus protects against infections and hemorrhages.

Use of thrombopoietic growth factors in acute leukemia.

Several hematopoietic growth factors have been shown to affect megakaryocyte development, and two, interleukin (IL)-11 and thrombopoietin (TPO) are presently being evaluated for use in patients with thrombocytopenia. In two studies patients who required one or more platelet transfusions during their first course of chemotherapy were found to require fewer platelet transfusions if their second cycle was augmented with IL-11. The drug was generally safe, with cardiovascular compromise the only significant complication occurring in a minority of patients. Although these reports included patients with various malignancies, studies of IL-11 in patients with myeloproliferative disorders have not been presented. In several clinical trials in cancer patients treatment with TPO was safe, and when administered early following a moderately aggressive cytotoxic insult was effective in accelerating platelet recovery. In addition, in both pre-clinical and clinical trials, TPO given to stem cell donors during mobilization lead to accelerated hematopoietic recovery. Finally, TPO appears safe when administered to patients with acute myelogenous leukemia (AML), both with respect to acute toxicity and long-term outcome of the leukemia. However, when used following a 7-day course of standard chemotherapy, the agent does not appear to accelerate platelet recovery. As such, additional clinical trials to test different growth factor regimens are ongoing. A number of studies have suggested that megakaryocytic growth factors may play a role in the biology of myeloproliferative disorders. Given the potential for adversely affecting patients with these disorders, the affects of IL-11 or TPO in patients with AML must continue to be carefully studied.

Megakaryocyte growth and development factor-induced proliferation and differentiation are regulated by the mitogen-activated protein kinase pathway in primitive cord blood hematopoietic progenitors.

In several erythroleukemia cell lines, activation of mitogen-activated protein kinases (MAPK) by phorbol esters or megakaryocyte growth and development factor (MGDF) is required for induction of megakaryocytic phenotype and growth arrest. To support this model, we have examined the effect of a specific inhibitor of this pathway (PD98059) on human CD34(+) hematopoietic progenitors isolated from cord blood (CB), induced to differentiate along the megakaryocytic lineage in liquid cultures supplemented with rhuMGDF. RhuMGDF induced a sustained activation of MAPK in megakaryocytes and this activation was completely inhibited in the presence of low concentrations of PD98059 (6 to 10 micromol/L). At this concentration, PD98059 induced an increase in cell proliferation, resulting in accumulation of viable cells and a prolongation of the life time of the cultures. This increase correlated with an increase in DNA synthesis rather than with a reduction in apoptosis. This effect was combined with developmental changes indicative of delayed megakaryocytic differentiation: (1) PD98059-treated cells tended to retain markers of immature progenitors as shown by the increased proportion of both CD34(+) and CD41(+)CD34(+) cells. (2) PD98059-treated cultures were greatly enriched in immature blasts cells. (3) PD98059 increased megakaryocytic progenitors able to form colonies in semisolid assays. Thus, the MAPK pathway, although not required for megakaryocyte formation, seems to be involved in the transition from proliferation to maturation in megakaryocytes. Inhibition of MAPK activation also led to an increase in the number and size of erythroid colonies without affecting granulocyte/macrophage progenitor numbers suggesting that, in addition to the megakaryocytic lineage, the MAPK pathway could play a role in erythroid lineage differentiation.

Memorial lecture. Megakaryocytic growth factors: is there a new approach for management of thrombocytopenia in patients with malignancies?

C-mpl ligand acts primarily as a lineage-specific hematopoietic growth factor by promoting proliferation of megakaryocyte precursors and their differentiation into megakaryocytes and platelets. In addition to the ability of c-mpl ligand to support megakaryocytic development from CD34+ precursor cells, several lines of evidence also point to a stimulatory effect on hematopoietic stem cells. When recombinant thrombopoietin or pegylated megakaryocyte growth and development factor is administered to normal animals or humans, there is a dose-dependent increase in the platelet count. When administered following chemotherapy in animal models or humans, c-mpl ligands reduce the duration and sometimes the degree of thrombocytopenia. The issue of whether clinically relevant thrombocytopenia can be ameliorated has so far been more difficult to resolve. Because severe thrombocytopenia is not commonly seen with standard chemotherapy regimens, clinical studies examining c-mpl ligands for their ability to ameliorate chemotherapy-induced thrombocytopenia will focus on treatment of acute leukemias and bone marrow transplantation. The potential utility of c-mpl ligands for treatment of myelodysplastic syndromes, aplastic anemias, or in HIV infection, will have to be evaluated in the future. Possibly the greatest potential of thrombopoietic growth factors in the near future may be in transfusion medicine, to collect and to store platelets from healthy donors or in autologous settings.

The correlation between erythropoiesis and thrombopoiesis as an index for pre-operative autologous blood donation.

We have previously reported that the platelet count and an increase in platelet number reflect individual erythropoietic capacity in pre-operative autologous blood donation (PABD). We have examined the correlation between erythropoiesis and thrombopoiesis by quantitative in vitro determination of thrombopoietin (TPO), erythropoietin (EPO), and interleukin-6 (IL-6) in patients with PABD. A sequential increase in platelet count with donation could not be explained by an increase in TPO. TPO showed a tendency to be inversely related to the pre-donation platelet count, and to be related to the pre-donation hemoglobin level. There was an inverse relationship between the TPO and EPO levels. As seen with these results, a high erythropoietic state induces restraint of thrombopoiesis, and a low erythropoietic state induces an increase in thrombopoiesis. These effects modulate EPO and TPO via negative feedback. These results provide some practical important information for performing autologous blood donation. Further studies are needed to elucidate the details of these correlations.

PEG-rHuMGDF promotes multilineage hematopoietic recovery in myelosuppressed mice.

PEG-rHuMGDF administered to normal mice is a lineage-specific growth factor for megakaryocytes and platelets as judged by morphologic examination of hematologic cells in marrow and peripheral blood smears. The purpose of this study was to document that PEG-rHuMGDF in myelosuppressed mice promotes multilineage hematopoietic recovery. High-dose 5-fluorouracil (5-FU) in mice results in profound myelosuppression and 0-30% survival. Mice receiving a single dose of PEG-rHuMGDF (1000 microg/kg) 1 day after 5-FU (225 mg/kg) demonstrate an increased survival (76% vs 27% in control mice at 14 days). Compared to surviving controls, PEG-rHuMGDF-treated mice not only show the expected higher platelet counts, but also increased marrow colony-forming unit granulocyte-macrophage, increased multilineage marrow cellularity, and increased neutrophil, monocyte, and lymphocyte counts in peripheral blood. PEG-rHuMGDF- and vehicle-treated mice both develop hepatic abscesses after 5-FU treatment, but the abscesses in the PEG-rHuMGDF-treated mice contain more neutrophils, suggesting that myeloid reconstitution contributes to their survival. Furthermore, survival in 5-FU-treated mice is significantly improved by granulocyte colony-stimulating factor and antibiotics, suggesting that infection rather than thrombocytopenia is the predominant cause of death. PEG-rHuMGDF after 5-FU promotes survival accompanied by accelerated lymphohematopoietic repopulation, suggesting that PEG-rHuMGDF, a lineage-specific thrombopoietic factor in normal mice, promotes multilineage hematopoietic recovery in myelosuppressed mice.

The efficacy of recombinant thrombopoietin in murine and nonhuman primate models for radiation-induced myelosuppression and stem cell transplantation.

Radiation-induced pancytopenia proved to be a suitable model system in mice and rhesus monkeys for studying thrombopoietin (TPO) target cell range and efficacy. TPO was highly effective in rhesus monkeys exposed to the mid-lethal dose of 5 Gy (300 kV x-rays) TBI, a model in which it alleviated thrombocytopenia, promoted red cell reconstitution, accelerated reconstitution of immature CD34+ bone marrow cells, and potentiated the response to growth factors such as GM-CSF and G-CSF. In contrast to the results in the 5 Gy TBI model, TPO was ineffective following transplantation of limited numbers of autologous bone marrow or highly purified stem cells in monkeys conditioned with 8 Gy TBI. In the 5 Gy model, a single dose of TPO augmented by GM-CSF 24 h after TBI was effective in preventing thrombocytopenia. The strong erythropoietic stimulation may result in iron depletion, and TPO treatment should be accompanied by monitoring of iron status. This preclinical evaluation thus identified TPO as a potential major therapeutic agent for counteracting radiation-induced pancytopenia and demonstrated pronounced stimulatory effects on the reconstitution of immature CD34+ hemopoietic cells with multilineage potential. The latter observation explains the potentiation of the hematopoietic responses to G-CSF and GM-CSF when administered concomitantly. It also predicts the effective use of TPO to accelerate reconstitution of immature hematopoietic cells as well as possible synergistic effects in vivo with various other growth factors acting on immature stem cells and their direct lineage-committed progeny. The finding that a single dose of TPO might be sufficient for a clinically significant response emphasizes its potency and is of practical relevance. The heterogeneity of the TPO response encountered in the various models used for evaluation points to multiple mechanisms operating on the TPO response and heterogeneity of its target cells. Mechanistic mouse studies made apparent that the response of multilineage cells shortly after TBI to a single administration of TPO is quantitatively more important for optimal efficacy than the lineage-restricted response obtained at later intervals after TBI and emphasized the importance of a relatively high dose of TPO to overcome initial c-mpl-mediated clearance. Further elucidation of mechanisms determining efficacy might very well result in a further improvement, e.g., following transplantation of limited numbers of stem cells. Adverse effects of TPO administration to myelosuppressed or stem cell transplanted experimental animals were not observed.

Terminal megakaryocytic differentiation of TF-1 cells is induced by phorbol esters and thrombopoietin and is blocked by expression of PML/RARalpha fusion protein.

We have analyzed the differentiation program of growth factor-dependent TF-1 erythroleukemia cells as well as clones with inducible expression of the APL-specific PML/RARalpha protein. We have shown that TF-1 cells may be induced to megakaryocytic differentiation by phorbol ester (phorbol dibutyrate, PDB) addition, particularly when combined with thrombopoietin (Tpo). RT-PCR studies showed that Tpo induces Tpo receptor (TpoR or c-mpl), whose expression was further potentiated by PDB addition. When the cells are induced with both PDB and Tpo erythropoietin receptor (EpoR) expression was inhibited. In the absence of Zn2+-induced PML/RARalpha expression, PDB and Tpo induced megakaryocytic differentiation of TF-1 MTPR clones as observed in 'wild-type' TF-1 cells. Conversely, when PML/RARalpha expression was induced by Zn2+, PDB and Tpo treatment of these clones caused only a reduced level of megakaryocytic differentiation. These observations indicate that: (1) TF-1 cells as well as other erythroleukemic cells, possess the capacity to differentiate to megakaryocytic cells when grown in the presence of protein kinase (PKC) activators and more efficiently when combined with Tpo; (2) the PML/RARalpha gene has a wide capacity to interfere with the program of hematopoietic differentiation, including megakaryocytic differentiation. Finally, we also observed that PML/RARalpha expression in TF-1 cells induces an up-modulation of interleukin-3 receptor, c-kit and c-mpl, a phenomenon which may offer these cells a growth advantage.

Multilineage hematopoietic recovery by a single injection of pegylated recombinant human megakaryocyte growth and development factor in myelosuppressed mice.

Previous studies have shown that daily multiple administration of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) markedly stimulates thrombopoiesis and effectively ameliorates thrombocytopenia, and in most cases anemia and neutropenia, in myelosuppressed animals. In this study, we evaluated the effects of a single intravenous injection of PEG-rHuMGDF on hematopoietic recovery after sublethal total-body irradiation in mice. A single injection of PEG-rHuMGDF (1 to 640 microg/kg) 1 hour after irradiation accelerated platelet, red blood cell (RBC), and white blood cell (WBC) recovery in a dose-dependent fashion. In the bone marrow of vehicle-treated mice, megakaryocytic, erythroid, and myeloid progenitors, as well as day 12 colony-forming unit-spleen (CFU-S), were dramatically decreased much earlier than the nadirs of peripheral blood cells, whereas megakaryocytes were modestly decreased. Treatment with PEG-rHuMGDF (80 microg/kg, an optimal dose) 1 hour after irradiation resulted in more rapid recovery of these four hematopoietic progenitors and also significantly facilitated megakaryocyte recovery. In addition, the same PEG-rHuMGDF administration schedule expanded bone marrow cells capable of rescuing lethally irradiated recipient mice. As the interval between irradiation and PEG-rHuMGDF treatment was longer, its effects on hematopoietic recovery were attenuated. In contrast to the effects of PEG-rHuMGDF, a single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 1 hour after irradiation exclusively accelerated WBC recovery, but only to a similar extent as PEG-rHuMGDF (80 microg/kg) treatment even when rhG-CSF doses were escalated to 1,000 microg/kg. This appeared related to different pharmacokinetics of these two factors after a single injection in irradiated mice. The concentrations of PEG-rHuMGDF after injection persisted in the plasma for a longer time compared with rhG-CSF. These results indicate that a single injection of PEG-rHuMGDF at an early time after irradiation is able to effectively improve thrombocytopenia, anemia, and leukopenia with concomitant accelerated recovery of both primitive and committed hematopoietic progenitors in irradiated mice. Our data also show that compared with the rhG-CSF shown to exert multilineage effects on hematopoiesis, PEG-rHuMGDF has more wide-ranging effects on peripheral blood cell recovery.