Megakaryocyte Growth And Development Factor Research Articles

Systematic literature review and meta-analysis on use of Thrombopoietic agents for chemotherapy-induced thrombocytopenia.

BackgroundCurrently, there are no approved options to prevent or treat chemotherapy-induced thrombocytopenia (CIT). We performed a systematic literature review and meta-analysis on use of thrombopoietic agents for CIT.Patients and methodsWe searched Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, PubMed, EMBASE, ClinicalTrials.gov, and health technology assessments from January 1995 to March 2021 for studies evaluating thrombopoietic agents for CIT, including recombinant human thrombopoietin (rhTPO), megakaryocyte growth and development factor (MGDF), romiplostim, and eltrombopag. Random effects meta-analyses were conducted for efficacy and safety endpoints.ResultsWe screened 1503 titles/abstracts, assessed 138 articles, and abstracted data from 39 publications (14 recombinant human thrombopoietin, 7 megakaryocyte growth and development factor, 9 romiplostim, 8 eltrombopag, and 1 romiplostim/eltrombopag). Random effects meta-analyses of data from multiple studies comparing thrombopoietic agents versus control (comparator, placebo, or no treatment) showed that thrombopoietic agents did not significantly improve chemotherapy dose delays and/or reductions (21.1% vs 40.4%, P = 0.364), grade 3/4 thrombocytopenia (39.3% vs 34.8%; P = 0.789), platelet transfusions (16.7% vs 31.7%, P = 0.111), grade ≥ 2 bleeding (6.7% vs 16.5%; P = 0.250), or thrombosis (7.6% vs 12.5%; P = 0.131). However, among individual studies comparing thrombopoietic agents with placebo or no treatment, thrombopoietic agents positively improved outcomes in some studies, including significantly increasing mean peak platelet counts (186 x 109/L with rhTPO vs 122 x 109/L with no treatment; P < 0.05) in one study and significantly increasing platelet count at nadir (56 x 109/L with rhTPO vs 28 x 109/L with not treatment; P < 0.05) in another study. Safety findings included thrombosis (n = 23 studies) and bleeding (n = 11), with no evidence of increased thrombosis risk with thrombopoietic agents.ConclusionOur analyses generate the hypothesis that thrombopoietic agents may benefit patients with CIT. Further studies with well-characterized bleeding and platelet thresholds are warranted to explore the possible benefits of thrombopoietic agents for CIT.

Thrombopoietin is associated with a prognosis of gastric adenocarcinoma.

OBJECTIVE Thrombopoietin (THPO) is well-known as a megakaryocyte growth and development factor (MGDF) involved in megakaryocyte proliferation and maturation. To explore the biological effects of THPO in gastric adenocarcinoma, we conducted this study. Methods: By accessing the TCGA database, the expression level of THPO was determined in tumor tissues. The association between THPO expression and clinical features, or prognostic significance was described by Cox regression analysis and Kaplan-Meier. The SiRNA method was used to decline the THPO expression; then cell viability, invasion, and migration were detected to verify the effects of the knockdown of THPO. qPCR and western blotting were implemented to examine the expression level of THPO. Results: The expression of THPO was increased in tumor tissue and cells, its high-regulation was associated with a poor prognosis in patients with gastric adenocarcinoma. Cell viability, invasion, and migration were suppressed in AGS with the down-regulation of THPO. Furthermore, on the basis of si-THPO transfection, E-cadherin was promoted while N-cadherin and Vimentin were attenuated. CONCLUSION Our results revealed that THPO may be a potent marker of gastric adenocarcinoma, providing a novel potential screening method for gastric adenocarcinoma.

A network map of thrombopoietin signaling.

Thrombopoietin (THPO), also known as megakaryocyte growth and development factor (MGDF), is a cytokine involved in the production of platelets. THPO is a glycoprotein produced by liver and kidney. It regulates the production of platelets by stimulating the differentiation and maturation of megakaryocyte progenitors. It acts as a ligand for MPL receptor, a member of the hematopoietic cytokine receptor superfamily and is essential for megakaryocyte maturation. THPO binding induces homodimerization of the receptor which results in activation of JAKSTAT and MAPK signaling cascades that subsequently control cellular proliferation, differentiation and other signaling events. Despite the importance of THPO signaling in various diseases and biological processes, a detailed signaling network of THPO is not available in any publicly available database. Therefore, in this study, we present a resource of signaling events induced by THPO that was manually curated from published literature on THPO. Our manual curation of thrombopoietin pathway resulted in identification of 48 molecular associations, 66 catalytic reactions, 100 gene regulation events, 19 protein translocation events and 43 activation/inhibition reactions that occur upon activation of thrombopoietin receptor by THPO. THPO signaling pathway is made available on NetPath, a freely available human signaling pathway resource developed previously by our group. We believe this resource will provide a platform for scientific community to accelerate further research in this area on potential therapeutic interventions.

1563P - Literature review of TPOR agonists for CIT

Background: Chemotherapy-induced thrombocytopenia (CIT) can lead to dose delay/reduction. Currently there are no specific treatment recommendations beyond transfusion. We performed a systematic literature search on the use of thrombopoietin receptor (TPOR) agonists for CIT. Methods: We searched the Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, PubMed, EMBASE, clinicaltrials.gov, and Health Technology Assessments from 1995-2017 for studies of TPOR agonists [eg, romiplostim, eltrombopag, TPO, and megakaryocyte growth and development factor (MGDF)] for CIT. Each publication was independently reviewed by two people and data extracted into Excel. Results: We screened 892 titles/abstracts, assessed 52 articles, and abstracted data from 14 articles and 15 abstracts/posters from 1997-2016 (10 TPO, 7 MGDF, 8 romiplostim, and 4 eltrombopag), which included 18 randomized trials. TPOR agonist regimens varied widely. Common cancers included leukemia/lymphoma (n = 8 studies) and non-small cell lung cancer (n = 4), and common chemotherapies were platinum-based (n = 15) or included cytarabine (n = 5). Median or mean baseline platelet counts were 56 × 109/L-324 × 109/L in studies to treat CIT (N = 7) and 109 × 109/L-597 × 109/L in studies to prevent CIT (N = 22). The 16 placebo-controlled or crossover studies (MGDF n = 6 studies, TPO n = 5, romiplostim n = 3, eltrombopag n = 2) generally found that TPOR agonists increased platelet counts and reduced transfusions and dose delays/reductions (Table). Safety measures included thromboses (n = 19 studies) and bleeding (n = 8).Table1563P Study Design and Endpoints TPOR Agonist vs. Control (Range study incidences)CIT Prevention: Vs. Placebo/Observation or Crossover (n = 16 studies)TPOR Agonist (N = 625)Control (N = 428)Efficacy EndpointChemotherapy dose delay/reduction3% - 40%58% - 75%Grade 3-4 thrombocytopenia0% - 100%0% - 42%Platelet transfusions6% - 58%8% - 83%Safety EndpointThrombosis0% - 29%0% - 33%Bleeding0% - 100%0% - 50%CIT Treatment: Vs. rhIL-11 (n = 2 studies)TPOR Agonist (N = 63)Control (N = 71)Efficacy EndpointChemotherapy dose delay/reductionN/AN/AGrade 3-4 thrombocytopeniaGrade 3: 54% Grade 4: 14%Grade 3: 85% Grade 4: 41%Platelet transfusions11%30%Safety EndpointThrombosisN/AN/ABleedingN/AN/ACIT Treatment: Vs. PBO/Observation or Crossover (n = 2 studies)TPOR Agonist (N = 172)Control (N = 65)Efficacy EndpointChemotherapy dose delay/reductionN/AN/AGrade 3-4 thrombocytopeniaN/AN/APlatelet transfusionsN/AN/ASafety EndpointThrombosis5% - 13%7% - 21%Bleeding2% - 9%9% Open table in a new tab Conclusions: While TPOR agonists have not been approved for use in CIT, this literature review suggests that TPOR agonists may increase platelet counts and decrease chemotherapy dose delay/reduction. Further study with well-characterized bleeding and platelet thresholds is needed to explore the possible benefits of TPOR agonists for CIT compared with current care options (eg, transfusions, dose reduction). Legal entity responsible for the study: Amgen Inc. Funding: Amgen Inc. Disclosure: G.A. Soff: Research support from Amgen. J. Fryzek: Employee of EpidStat, which serves as a consultant with Amgen. M. Mullins: Consultant for EpidStat, which itself consults for Amgen. L.C. Bylsma: Employee of EpidStat, which consults for Amgen. J.K. Park: Amgen employee. All other authors have declared no conflicts of interest.

Clinical-Scale Cultures of Cord Blood CD34+ Cells to Amplify Committed Progenitors and Maintain Stem Cell Activity

We developed a clinical-scale cord blood (CB) cell ex vivo procedure to enable an extensive expansion of committed progenitors--colony-forming cells (CFCs) without impairing very primitive hematopoietic stem cells (HSCs). CD34(++) cells, selected from previously cryopreserved and thawed CB units, were cultured in two steps (diluted 1:4 after 6 days) in the presence of stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt-3L), megakaryocyte growth and development factor (MGDF) (100 ng/ml each), granulocyte-colony stimulating factor (G-CSF) (10 ng/ml) in HP01 serum-free medium. HSC activity was evaluated in a serial transplantation assay, by detection of human cells (CD45, CD33, CD19 and CFC of human origin) in bone marrow (BM) of primary and secondary recipient NOD/SCID mice 6-8 weeks after transplantation. A wide amplification of total cells (∼350-fold), CD34(+) cells (∼100-fold), and CFC (∼130-fold) without impairing the HSC activity was obtained. The activity of a particular HSC subpopulation (SRC(CFC)) was even enhanced.Thus, an extensive ex vivo expansion of CFCs is feasible without impairing the activity of HSCs. This result was enabled by associating antioxidant power of medium with an appropriate cytokine cocktail (i.e., mimicking physiologic effects of a weak oxygenation in hematopoietic environment).

Cytokines for the Treatment of Thrombocytopenia

Multiple cytokines affect the cellular processes that occur during the transition of a hematopoietic stem cell (HSC) to a platelet. Thrombopoietin (TPO) is the physiological regulator of thrombopoiesis. Although a number of cytokines (interleukin [IL]-1, IL-3, and IL-6) were first evaluated for their ability to lessen the degree of thrombocytopenia occurring during a variety of clinical scenarios, their clinical development was abandoned due to their limited effectiveness or excessive toxicity. Clinical results with TPO and a truncated pegylated form of TPO, megakaryocyte growth and development factor (MGDF), were more promising, but the repeated use of MGDF resulted in the development of neutralizing antibodies. This adverse event halted the further clinical development of not only MGDF but also TPO. IL-11 also affects various stages of megakaryocytopoiesis and thrombopoiesis and its use has been shown to shorten the duration of chemotherapy-induced thrombocytopenia, which led to its approval by the US Food and Drug Administration (FDA). A growing number of new non-immunogenic peptides and non-peptide TPO agonists recently have entered clinical trials. These small molecules appear to be effective therapies and have acceptable toxicity, but additional clinical evaluation will be required prior to their approval for clinical use.

Prolonged High-Level Detection of Retrovirally Marked Hematopoietic Cells in Nonhuman Primates after Transduction of CD34+ Progenitors Using Clinically Feasible Methods.

Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell(HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells(STR), or the fibronectin fragment CH-296(FN), and various cytokines such as flt3 ligand(FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor(SCF), and FLT(our best cytokine combination in prior studies)with a combination of megakaryocyte growth and development factor(MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10–15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.

Large‐scale expansion and transplantation of CD34+ hematopoietic cells: in vitro and in vivo confirmation of neutropenia abrogation related to the expansion process without impairment of the long‐term engraftment capacity

Herein are reported the results obtained in all multiple myeloma patients transplanted with peripheral blood hematopoietic progenitor cells submitted to ex vivo expansion. Patients had blood progenitor cell mobilization with cyclophosphamide and filgrastim. CD34+ cells were expanded for 10 days in a medium containing granulocyte-colony-stimulating factor (G-CSF), stem cell factor, and megakaryocyte growth and development factor (MGDF). Twenty-seven patients underwent transplantation with expanded and nonexpanded cells and 7 patients underwent transplantation with expanded cells only. The median fold cell expansion was 29.1. The number of colony-forming unit-granulocyte-macrophage (CFU-GM) and CD34+ cells, and the long-term culture-initiating cell (LTC-IC) activity increased with median fold values of 14.7, 2.75, and 2.25, respectively. Postmyeloablative neutropenia was abrogated in 24 of 27 patients transplanted with expanded cells plus nonexpanded cells. The median duration of severe neutropenia was 0 days and correlated with the number of cells and CFU-GM infused. Survival was similar to that of a historical control group. Our LTC-IC and NOD-SCID mice studies showed that the expanded cells are able of sustaining long-term hematopoiesis. Seven other patients received transplantation with expanded cells alone. Absolute neutropenia was abrogated in 6 patients. The median duration of neutropenia was 0 days. Two patients who received the lower number of total cells or CFU-GM had brief secondary neutropenia, which resolved after G-CSF injections. CD34+ cells expanded ex vivo can abrogate absolute and severe neutropenia after high-dose therapy. The results of the amplification process are strongly related to the delay of hematopoietic recovery.

The Expansion of Megakaryocyte Progenitors from CD34+-Enriched Mobilized Peripheral Blood Stem Cells Is Inhibited by Flt3-L

This study aimed to determine the optimal growth factor combination for expansion of megakaryocyte (Mk) progenitors with clonogenic potential from CD34+-enriched mobilized peripheral blood stem cells (PBSC). Mobilized PBSC were monocyte depleted and CD34+ enriched, then cultured with various combinations of interleukin-3 (IL-3), IL-6, IL-11, Flt3 ligand (Flt3-L), stem cell factor (SCF), granulocyte-macrophage colonystimulating factor (GM-CSF), and erythropoietin (EPO), using a 2(7-3) IV fractional factorial design. Expansion of Mk committed progenitors (CD41+) and primitive precursors (CD61+ CD34+) was determined using FACS and colony-forming assays. Amplification of Mk progenitor production was attributed to IL-3 (p < 0.002), SCF (p < 0.001), and GM-CSF (p < 0.05). Flt3-L inhibited the production of total CD61+ cells (p < 0.05), CD61+CD34+ cells (p < 0.03), and total CD41a+ cells (p < 0.01). Addition of Flt3-L to the optimum growth factor combination of megakaryocyte growth and development factor (MGDF), SCF, IL-3, and GM-CSF caused the greatest increase in total nucleated cells but reduced Mk progenitor expansion. There was also a 20% reduction in Mk+ colonies from cells expanded in the presence of Flt3-L. Factorial analysis identified the optimal combination of growth factors required to expand Mk precursors with clonogenic potential. The addition of Flt3-L to the optimal combination of MGDF, SCF, IL-3, and GM-CSF reduced both the fold expansion of Mk progenitors and Mk colony numbers.

Megakaryocyte growth and development factor is a potent growth factor for primitive hematopoietic progenitors in the human fetus.

Megakaryocyte growth and development factor (MGDF), or thrombopoietin, has received considerable attention as a therapeutic agent for treating thrombocytopenia or for its use in the ex vivo culture of hematopoietic stem cells. MGDF is known to support the growth of a broad spectrum of hematopoietic precursors obtained from adult or neonatal tissues, but its effects on the growth of fetal progenitors and stem cells has not been studied. Human CD38(+)CD34(2+) progenitors and CD38(-)CD34(2+) cells, a population that contains stem cells, were isolated from midgestation liver and grown under defined conditions with MGDF and various cytokines known to support the growth of primitive hematopoietic precursors. In clonal assays of colony-forming cells (CFCs), MGDF supported the growth of 15-25% of candidate stem cells when combined with granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), flk-2/flt3 ligand, or stem cell factor. MGDF was observed to strongly support the early stages of hematopoiesis and expansion of high proliferative potential CFCs. More mature progenitors were expanded nearly 78-fold in 1 wk of culture with MGDF+SCF+GM-CSF. MGDF alone was also found to support the short-term (2 d) survival of CD38(-)CD34(2+) high proliferative potential CFCs. The effects of MGDF were more modest on CD38(+)CD34(2+) progenitors with only additive increases in colony formation being observed. These findings suggest that MGDF administration in fetuses and neonates may strongly affect the growth and mobilization of primitive hematopoietic progenitors and that MGDF may find use in the ex vivo growth and expansion of fetal stem cells.

Proliferation of human progenitor cells in a long-term culture system is more efficiently sustained by the addition of Flt-3 ligand or megakaryocyte growth and development factor than by Kit ligand.

We compared the effects of the early-acting growth factors (GF), Flt-3 ligand (FL), c-Kit ligand (KL), and leukemia inhibitory factor (LIF), and the late-acting GF, granulocyte-colony stimulating factor (G-CSF) and megakaryocyte growth and development factor (MGDF), added alone in human long-term marrow culture (LTMC). The GF were used in primary cultures of mononuclear cells (MNC) and in cocultures of CD34+ cells on murine preestablished MS-5 stromal layers. GF activity was assessed as nonadherent and adherent progenitor cell production and cobblestone area formation at week 5. In this system, only FL, KL, and MGDF significantly stimulated early stages of hematopoiesis, whereas only G-CSF stimulated the proliferation of mature progenitor cells within the granulo-monocyte lineage and no effect was observed with LIF. FL displayed the strongest activity, and MGDF was more efficient than KL, both in primary cultures of MNC and in cocultures of CD34+ cells. However, the stimulatory effects of these GF used alone were dependent on the presence of a stromal layer. These LTMC data emphasize the particular roles for FL and MGDF in the stimulation of primitive hematopoiesis.

In vitro response of myelodysplastic megakaryocytopoiesis to megakaryocyte growth and development factor (MGDF).

To evaluate a potential therapeutic role for megakaryocyte growth and development factor (MGDF) in myelodysplastic syndromes (MDS) we compared the in vitro response of normal and myelodysplastic megakaryopoiesis to MGDF on bone marrow mononuclear cells from nine MDS patients and four healthy donors in a short term liquid culture system. The cells were incubated with MGDF alone (1 ng/ml, 10 ng/ml and 100 ng/ml) and in combination with 20 ng/ml stem cell factor, 2 U/ml erythropoietin (EPO) and 100 ng/ml interleukin-3 (IL-3). Cytospins were prepared after 4 days and 10 days for CD61 APAAP staining. In addition, the ploidy of propidium iodide stained CD61 positive cells were detected by flow cytometry. The CD61+ cell number significantly increased (13-fold after 4 days, 56-fold after 10 days of culture) when MGDF (100 ng/ml) was added to the normal bone marrow (NBM) cultures (P=0.0003; P=0.0001). In the MDS cultures the absolute number of endomitotic cells as well as the percentage of polyploid cells remained significantly lower than in NBM after 10 days (P=0.0001). The effect of MGDF on polyploidization of MDS cells was significantly dose dependent (P=0.0051). We found no correlation between peripheral platelet count, cellularity of the bone marrow, number of bone marrow megakaryocytes or FAB-subtype and response to MGDF. Additional administration of IL-3 resulted in a left-shift of megakaryopoiesis in both groups. Even though the response of myelodysplastic megakaryocytic progenitors to MGDF shows inter-individual variations, it is significantly impaired overall. Our data suggest that higher doses of MGDF may be able to ameliorate thrombocytopenia in a subgroup of MDS patients.

Hematopoietic potential of murine skeletal muscle-derived CD45(-)Sca-1(+)c-kit(-) cells.

Somatic stem cells, which are poorly defined in postnatal mammalian tissues, are attractive candidates for examination of stem cell plasticity. Our goal was to determine the identity of neonatal muscle-derived cells that contain hematopoietic potential and to explore the status of CD45 expression on these cells. Skeletal muscle from thighs of 4- to 7-day-old mice was harvested, enzymatically digested, and flow cytometrically sorted to yield CD45(-)Sca-1(+)c-kit(-) cells. These cells were examined in hematopoietic colony-forming assays and competitive repopulation assays, and were expanded ex vivo. Additionally, CD45, c-kit, PU.1, and beta globin major expression was tracked over time in cultured cells to assess the possibility of manipulating stem cell differentiation in vitro. Freshly isolated CD45(-)Sca-1(+)c-kit(-) cells were devoid of hematopoietic lineage markers and contained no colony-forming activity but displayed superior long-term competitive repopulating ability when compared to freshly isolated muscle-derived CD45(+)Sca-1(+)c-kit(+) cells. CD45(-)Sca-1(+)c-kit(-) cells expanded ex vivo in 5 ng/mL murine stem cell factor, mFlt-3L, and megakaryocyte growth and development factor (MGDF) for 9 days increased their in vivo hematopoietic repopulating potential 5.3-fold relative to fresh cells. Although fresh cells did not transcribe mRNA of several hematopoietic genes, a small fraction of cells cultured for 9 days acquired cell surface c-kit, and only these cells expressed c-kit and PU.1 mRNA and maintained competitive repopulating ability, suggesting at least myeloid and perhaps lymphoid developmental potential. Neonatal murine muscle-derived cells expressing the phenotype CD45(-)Sca-1(+) c-kit(-) are putative adult somatic stem cells with in vitro and in vivo hematopoietic differentiation potential.

Enhanced expansion and maturation of megakaryocytic progenitors by fibronectin.

Megakaryopoiesis occurs as a result of a complex interaction between hemopoietic cells, stromal cells, hemopoietic growth factors and extracellular matrix (ECM). Megakaryocyte growth and development factor (MGDF) or thrombopoietin is the main growth factor that induces megakaryocyte commitment. The role of adhesion proteins in the generation of megakaryocytic progenitors has not been well studied. We isolated CD34(+) cells from umbilical cord (UC) blood and cultured them in serum-free medium containing IL-3, IL-6, SCF and MGDF, with or without fibronectin. Cultures were sampled at Days 4 and 8 for cell count, immunophenotyping by flow cytometry, and megakaryocyte colony-forming units (CFU-Mk) assay. Fibronectin in synergy with MGDF increased both total and CD34(+) cell count. Immunophenotyping of the CD34(+) and CD34(-) cells showed that the percentage expression of CD61 and CD41a on CD34(-) cells was increased by fibronectin compared with CD34(+) cells by Day 8 of culture. The CFU-Mk assay confirmed that fibronectin increased generation of megakaryocytic progenitors by 2.4-fold by Day 8 of culture, but the absolute number was less than that of Day 4 culture. The cells in the CFU-Mk colonies derived from culture containing fibronectin were larger than those from cytokine-only culture. It was concluded that the addition of fibronectin to the culture system enhanced MGDF, induced ex vivo expansion of megakaryocytic progenitors from CD34(+) cells, as well as increasing their maturation towards later stages of megakaryocyte differentiation.

Signaling by the Mpl receptor involves IKK and NF-kappaB.

Binding of tumor necrosis factor-alpha (TNF-alpha) to its receptor activates IKK complex, which leads to inducement of NF-kappaB activity. Here we report that activation of Mpl ligand is also linked to IKK and NF-kappaB activity. Mpl ligand, also known as thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF), induces megakaryocyte differentiation and inhibition of mitotic proliferation, followed by induction of polyploidization and fragmentation into platelets. The latter process is often observed in megakaryocytes undergoing apoptosis. Treatment of a Mpl ligand-responding megakaryocytic cell line with this cytokine led to an immediate, transient increase in IKK activity followed by a profound decrease in this kinase activity over time. This decrease was not due to an effect on the levels of the IKK regulatory components IKKalpha and IKKbeta. Proliferating megakaryocytes displayed a constitutive DNA-binding activity of NF-kappaB p50 homodimers and of NF-kappaB p50-p65 heterodimers. As expected, reduced IKK activity in Mpl ligand-treated cells was associated with a significant reduction in NF-kappaB DNA binding activity and in the activity of a NF-kappaB-dependent promoter. Our study is thus the first to identify a constitutive NF-kappaB activity in proliferating megakaryocytes as well as to describe a link between Mpl receptor signaling and IKK and NF-kappaB activities. Since a variety of proliferation-promoting genes and anti-apoptotic mechanisms are activated by NF-kappaB, retaining its low levels would be one potential mechanism by which inhibition of mitotic proliferation is maintained and apoptosis is promoted during late megakaryopoiesis.

CD133+ cell selection is an alternative to CD34+ cell selection for ex vivo expansion of hematopoietic stem cells.

CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. This study compared the expansion capacities of CD133(+) and CD34(+) cells isolated from the same cord blood (CB) samples. After 14 days culture in stroma-free, serum-free medium in the presence of stem cell factor (SCF), Flt3-1, megakaryocyte growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF), the CD133(+) and CD34(+) fractions displayed comparable expansion of the myeloid compartment (CFC, LTC-IC, and E-LTC-IC). The expansion of CD133(+) CB cells was up to 1262-fold for total cells, 99-fold for CD34(+) cells, 109-fold for CD34(+) CD133(+) cells, 133-fold for CFU-GM, 14.5-fold for LTC-IC, and 7.5-fold for E-LTC-IC. Moreover, the expanded population was able to generate lymphoid B (CD19(+)), NK (CD56(+)), and T (CD4(+) CD8(+)) cells in liquid or fetal thymic organ cultures, while expression of the homing antigen CXCR4 was similar on expanded and nonexpanded CD133(+) or CD34(+) cells. Thus, the CD133(+) subset could be expanded in the same manner as the CD34(+) subset and conserved its multilineage capacity, which would support the relevance of CD133 for clinical hematopoietic selection.

Platelets Exposed to Elevated Levels of Endogenous Thrombopoietin In Vivo Have a Reduced Response to Megakaryocyte Growth and Development Factor In Vitro

Thrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF), has been shown to potentiate the sensitivity of normal human platelets to various agonists in vitro. The present study investigated the functional and biochemical properties of platelets from mice rendered thrombocytopenic by sublethal irradiation with regard to the reactivity to recombinant murine MGDF (rmMGDF) in vitro. During the course of reversible thrombocytopenia following irradiation, platelets from irradiated mice which had lower platelet counts and reciprocally higher plasma TPO levels showed lower reactivity to rmMGDF in agonist-induced platelet aggregation. Intravenous injections of recombinant soluble murine c-Mpl (sMpl), which has the ability to capture TPO, after irradiation restored the reactivity of platelets at the platelet nadir to rmMGDF. On the other hand, platelets prepared from normal mice 3 h after a single intravenous injection of pegylated rmMGDF did not respond to rmMGDF. There was a marked decrease in c-Mpl and Janus kinase 2 (JAK2) in platelets from irradiated mice at the platelet nadir. Similar results were observed with platelets from mice administered pegylated rmMGDF. JAK2 was only moderately decreased, however, in platelets from mice given sMpl after irradiation. These results indicate that exposure of platelets to increased endogenous TPO levels in vivo in thrombocytopenic mice leads to a reduction in the platelet reactivity to rmMGDF in vitro. Further, these results suggest that the c-Mpl-mediated signaling pathway, which is essential for the priming effect of rmMGDF, is defective in thrombocytopenic murine platelets.

Characterization of cytokine interactions by flow cytometry and factorial analysis

Background Multiple cytokines are required for the growth and development of hematopoietic cells. The effect of many cytokines depends on the activity of other signaling pathways. These interactions are quantified using factorial experimental design and analysis. Methods Human umbilical cord blood (HUCB) CD34+ cells were cultured in fully defined media containing various combinations of recombinant cytokines as defined by resolution IV factorial (2) or full factorial (24) design experiments. The cytokines studied were stem cell factor (SCF), interleukin (IL)-3, megakaryocyte growth and development factor (MGDF), granulocyte-colony stimulating factor (G-CSF), Flt-3 ligand, IL-6, IL-11, and erythropoietin (EPO). In vitro cell divisions were tracked by staining CD34+ cells with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, followed by flow cytometric analysis at 4 days of culture. In separate experiments, lineage commitment and differentiation were determined at 7 days by immunophenotype. Results In addition to the main effects of single cytokines, cytokine interactions were identified. There was a negative interaction between IL-3 and MGDF that resulted in a less than additive effect of these factors on erythroid and megakaryocytic development. The effect of Flt-3 ligand and SCF factor on CD34+ cell production was also less than additive, although the response to both cytokines was greater than single cytokines. The only positive interaction that was identified was between EPO and SCF, which resulted in the synergistic production of erythroid cells. Conclusions Factorial analysis provides a powerful methodology to study the integration of multiple signals at the cellular and molecular level. Cytometry 43:69–81, 2001. © 2001 Wiley-Liss, Inc.

Adenoviral vector design for high-level transgene expression in primitive human hematopoietic progenitors.

Adenoviral vector-mediated transient gene expression can provide new possibilities for ex vivo manipulation of quiescent hematopoietic stem cells (HSC). In order to define a suitable expression cassette for high levels of transgene expression in HSCs, we have studied the level of transgene expression in human CD34+CD38- cells using adenoviral vectors with various gene expression cassettes encoding the enhanced green fluorescence protein (EGFP) gene. CD34+ hematopoietic cells were cultured in serum-free medium with megakaryocyte growth and development factor (MGDF) alone for supporting the survival of primitive progenitors or with MGDF, c-kit ligand (KL) and flt3 ligand (FL) for inducing proliferation of primitive progenitors. With all the vectors tested, higher percentages of EGFP expressing cells were found in CD34+CD38- cells than those in CD34+CD38high cells from all donors tested. The phosphoglycerate kinase (PGK)-1 promoter was found to allow higher levels of EGFP expression than the human cytomegalovirus (HCMV) promoter in CD34+CD38- cells. Replacing the SV40 polyadenylation signal with the human beta-globin gene IVS2 and polyadenylation signal in the expression cassette (Ad5xPGK-EGFP-beta-globin) enhanced the level of EGFP expression markedly further. These results provide a guideline for the development of adenoviral vectors for gene expression in human primitive hematopoietic progenitor cells. Gene Therapy (2000) 7, 2132-2138.

Hypersensitivity of circulating progenitor cells to megakaryocyte growth and development factor (PEG-rHu MGDF) in essential thrombocythemia

Hematopoietic progenitor cells in 2 myeloproliferative disorders, juvenile chronic myelomonocytic leukemia and polycythemia vera, are known to be hypersensitive to cytokines that control normal progenitor cell proliferation, differentiation, and survival in their respective granulocyte/macrophage and erythroid lineages. Because thrombopoietin controls these functions in the normal megakaryocytic lineage, we asked the question: Are megakaryocytic progenitor cells in the myeloproliferative disorder essential thrombocythemia (ET) hypersensitive to thrombopoietin? Peripheral blood mononuclear cells from patients with ET, or secondary (reactive) thrombocytosis (2 degrees T), or healthy volunteers were grown in strictly serum-free agarose culture containing interleukin 3 (IL-3) and all-trans-retinoic acid, with various concentrations of PEG-rHu megakaryocyte growth and development factor (MGDF). The concentration of cytokine at half-maximum colony number served as a measure of progenitor cell sensitivity. Hypersensitivity to PEG-rHu MGDF was found in circulating progenitors from 18 of 20 (90%) informative patients with presumptive diagnosis ET, 1 of 8 (12.5%) 2 degrees T patients, and none of the 22 healthy volunteers. Median MGDF sensitivity ratio in ET patients was approximately 53 times greater than in the controls. This hypersensitivity, which was also directed to rHu thrombopoietin, was highly specific with respect to cytokine, disease, and cell lineage. We propose that, despite their single pluripotential cell origin, the different clinicopathologic phenotypes in different chronic myeloproliferative disorders are determined by lineage-restricted hypersensitivities of hematopoietic progenitor cells to endogenous cytokines. This work emphasizes the importance of stringent serum-free conditions for revealing true sensitivities to cytokines. The findings also offer a basis for evolving a positive test for ET, a diagnosis now made essentially by exclusion.