Megakaryocyte Colony Research Articles

Conditional Expression of C/EBPα in Mice Reveals Developmental Plasticity of Lymphoid and Erythroid/ Megakaryocyte Precursors in Vivo.

CCAAT/ enhancer binding protein α (C/EBP α) is a member of the bZIP family of transcription factors that plays a critical role for early myeloid differentiation. C/EBP α knockout mice showed a complete differentiation block at myeloblast stage in hematopoietic system, and mature neutrophils and eosinophils are absent in the peripheral blood. Lineage specification in developmental tree of hematopoiesis is generally determined by a lineage specific transcription factors such as C/EBP α and GATA-1 that allow commitment to the specific lineage with simultaneous extinction of their capacity to differentiate into the other ones. However, recent evidences shown by the ectopic expression of above transcription factors unveiled the unexpected developmental plasticity of various progenitors such as MEP (erythroid/ megakaryocyte progenitor) and CLP (common lymphoid progenitor). GATA-1 is reported to convert CLP and CMP (common myeloid progenitor) into erythroid/ megakaryocyte lineage, however, the effect of C/EBP α on MEP and CLP is still unlcear. In order to investigate the role of C/EBP α in the various aspect of hematopoietic differentiation, especially its effect on the lineage specification at different stages of differentiation in vivo, we generated transgenic mice expressing inducible form of C/EBP α (C/EBP α-ER) under H-2K promoter (C/EBP α-ER Tg). In these mice, C/EBP α activity can be induced conditionally by 4-hydroxy tamoxifen (4-HT) in all hematopoietic cells. As expected, C/EBP α-ER was expressed in almost all hematopoietic tissues including bone marrow, spleen and thymus in these mice. Gel shift analysis revealed that C/EBP α-ER was activated by 4-HT, and showed specific binding to C/EBP-specific oligonucleotide in these tissues. Next we tested differentiation plasticity of erythroid and lymphoid progenitors by ectopically inducing C/EBP α-ER activity in these cells. We sorted MEP and CLP by FACS from C/EBP α-ER Tg and examined their clonogenic activities in the presence or absence of 4-HT. In the absence of 4-HT, MEP and CLP exclusively formed erythroid/ megakaryocyte and lymphoid colonies, respectively, as previously reported. Surprisingly however, these cells dramatically changed their fate of differentiation and formed significant numbers of granulocyte/ macrophage (GM) colonies in the presence of 4-HT, indicating that ectopic activation of C/EBP α-ER activity skewed their differentiation pathways to myeloid lineage. Cytospin preparation of the colonies and RT-PCR analysis revealed that these were accompanied by the morphological differentiation to granulocytes/ macrophages, and upregulation of myeloid-specific genes at mRNA level. These results indicate that MEP and CLP are not fully committed to either erythroid/ megakaryocyte or lymphoid lineage, and possess differentiation plasticity that can be redirected to myeloid lineage.

SDF-1/CXCL12 Enhances Survival of Murine Embryonic Stem Cells and Their Production of Myeloid Progenitors, but Not Hemangioblasts.

Stromal cell derived factor (SDF)-1/CXCL12 has been shown to promote the survival of embryonic retinal ganglion cells, peritoneal B1a (PerBla) lymphocytes and chronic lymphocytic leukemia B cells. Our previous studies implicated SDF-1 as an important factor in enhancing survival of murine bone marrow (BM) hematopoietic stem cells and, human cord blood and adult human BM myeloid progenitors. Since pluripotent embryonic stem (ES) cells can give rise to differentiated cell types derived from all three primary germ layers (endoderm, mesoderm, and ectoderm) and adult stem cells are generated during embryoid body (EB) formation, we investigated whether SDF-1 has effects on survival of ES cells and EB generation of hematopoietic progenitor cells. In order to establish SDF-1 expression patterns during EB formation, we screened supernatants during day 1–5 EB formation for SDF-1 production by three murine ES cell lines (E14, R1 and CCE). We observed low but detectable SDF-1 secreted in cultures of ES cells and day 1 stage EBs. SDF-1 was increased in the media from day 2 stage EBs and continued to increase through days 4–5. CXCR4, SDF-1 receptor, expression was also analyzed. CXCR4 mRNA expression was low in ES cells and day 1–3 EBs, and increased significantly from Day 4 EBs, reaching maximum levels at day 5, and decreasing after day 6. Surface CXCR4 expression was consistent with mRNA data. To determine if SDF-1 had an effect on ES cell survival, we cultured ES cells without serum, and added serum at either 0, 24, 48 or 96 hrs to each of the following groups: A) Control, B) SDF-1 (100ng/ml) or C) AMD3100 (1 μM), an SDF-1 receptor (CXCR4) antagonist. Colonies were scored 7 days after the addition of serum. SDF-1 enhanced survival of ES cells, while AMD3100 decreased survival. We also checked the apoptosis of ES cells after withdrawing serum for 24, 48, 72 and 96 hours in four groups: A) control, B) SDF-1 (100ng/ml), C) AMD3100 (1 μM) or D) AMD3100 (1 μM) and SDF-1 (100ng/ml). SDF-1 decreased apoptosis and AMD3100 blocked the SDF-1 effect. AMD3100 alone increased apoptosis compared to control. This suggests that AMD3100 blocked endogenous SDF-1 effects. To determine if SDF-1 had an effect on differentiation of hematopoietic progenitor cells, we added SDF-1 (100 ng/ml), AMD3100, or AMD3100 plus SDF-1 (100 ng/ml) at the beginning of EB formation, immediately after removal of LIF, and quantitated primitive erythroid (p-BFU-E), definitive erythroid (d-BFU-E), granulocyte-macrophage (CFU-GM) and multipotential Granulocyte/Erythroid/Macrophage/Megakaryocyte (CFU-GEMM) colony formation. In comparison to control cells (cultured without SDF-1 and AMD3100), SDF-1 increased numbers of p-BFU-E, d-BFU-E, CFU-GM, and CFU-GEMM colonies. Addition of AMD3100 with SDF-1 blocked the enhancing effect of SDF-1. In addition, significantly decreased numbers of colonies were also observed in the presence of AMD3100 alone. This suggests that AMD3100 blocks endogenous SDF-1 actions, consistent with our data on SDF-1 production during EB formation. In order to determine when SDF-1 starts affecting hematopoiesis, hemangioblast colony assays were used. Neither SDF-1 nor AMD3100 influenced hemangioblast colony formation or expression of Flk-1 mRNA, a marker of hemangioblasts. The results suggest a role for SDF-1 in ES cell growth and differentiation.

Hematopoiesis in the Ts65Dn Mouse Model of Down Syndrome (DS) Recapitulates Many Features of Human DS.

Down syndrome-associated acute megakaryocytic leukemia (DS-AMKL) is a complex malignancy that evolves in hematopoietic progenitors with trisomy 21 that acquire a somatic mutation in the blood transcription factor GATA1. The mechanistic relationship between these two genetic factors that leads to leukemia is poorly understood. In order to study the interplay between trisomy 21 and GATA1 mutations, we are developing a mouse model of this malignancy. The Ts65Dn mouse, which contains a segmental trisomy for mouse chromosome 16, homologous to human 21, has been reported to display several of the cognitive and craniofacial phenotypes seen in humans with DS, but the hematopoietic system has not been assessed as a model for blood development in humans with DS. We have evaluated adult hematopoiesis in the Ts65Dn strain by comparing monthly complete blood counts (CBC) of peripheral blood from 14 trisomic and 20 disomic littermates. Similar to humans with DS, Ts65Dn trisomic mice display persistent erythrocyte macrocytosis, with values at the high end of the normal range. Trisomic mice also harbor decreased numbers of red blood cells, mildly elevated platelet counts, a higher percentage of monocytes and a lower hemoglobin concentration. Interestingly infants with DS frequently display thrombocytosis. In addition, we have characterized fetal liver hematopoiesis in the Ts65Dn strain by FACS analysis of hematopoietic precursors and by performing colony assays. In general, we did not detect any significant differences in erythroid, myeloid, or megakaryocytic colony formation between trisomic or disomic fetuses. Likewise flow cytometry for CD34, TER119, and CD41 demonstrated overall similar numbers of cells in these compartments for Ts65Dn mice and disomic littermates. However, one of seven trisomic embryos displayed a significant increase in the proportion of CD34+ cells with concomitant decrease in both Ter119+ and CD41+ populations. In addition, cells from this fetal liver gave rise to seven-fold and three-fold increases in BFU-E and CFU-Mk colonies respectively, with no change in the CFU-GM. Although the sample size is small, these findings suggest that a subset of Ts65Dn trisomic fetuses exhibit aberrant hematopoiesis. Taken together, our study indicates that the Ts65Dn trisomic mouse may be an excellent model to study human DS hematopoiesis.

Mice with AML1 (Runx1) Haploinsufficiency Have Significant Platelet Abnormalities That Accurately Mimic Those Seen in Human Patients with Germ Line Inactivating Mutations in AML1.

The AML1/CBFβ transcription complex, a critical regulator of the formation of definitive hematopoietic stem cells (HSC), is one of the most frequent targets of genetic alterations in acute leukemia. In addition to somatic alterations of AML1 and CBFβ in acute leukemia, germ-line loss-of-function mutations of AML1 are the underlying cause of an autosomal dominant familial platelet disorder with a predisposition to acute myeloid leukemia (FPD/AML). Importantly, a subset of the mutations identified in families with FPD/AML result in AML1 null allele, suggesting that AML1 haploinsufficiency is the underlying molecular abnormality. To explore the functional consequences of AML1 halpoinsufficiency on megakaryocyte development and platelet function, we analyzed the hematopoietic system of AML1+/- mice. Loss of a single AML1 allele resulted in a 15% reduction in the number of circulating platelets and a significant impairment in platelet function including a decrease in dense granule content and an impaired ability to aggregate in response to collagen stimulation. Further analysis indentified a left shift in the DNA ploidy of megakaryocytes and a reduction in GPV expression, consistent with impaired megakaryocyte maturation. In addition, electron microscopy indicated a reduction in platelet demarcation channels within the cytoplasm of megakaryocytes. Importantly, however, we did not observe a reduction in the total number of megakaryoctyes or a decrease in megakaryocyte colony forming units. These data suggest that the haploinsufficiency of AML1+/− does not alter the initial formation of megakaryocytes, but instead impairs the ability of these cells to efficiently mature and produce functional platelets. To explore the underlying mechanism responsible for the observed impairment in megakaryocyte maturation, we analyzed the pattern of expression of several putative AML1 transcriptional targets. Although AML1 binding sites have been identified within the promoter of c-mpl, the gene encoding the receptor for thrombopoietin (TPO), we did not observe any difference in c-mpl expression levels or in circulating TPO concentration between AML1+/− and +/+ mice. In addition, in vivo TPO stimulation induced a similar magnitude of megakaryocyte maturation and platelet production in both AML1+/+ and +/− mice. By contrast, analysis of members of the protein kinase C (PKC) family of gene, several which have been identified as transcriptional targets of AML1, revealed a reduction in PKCδ levels in platelets from AML1+/− mice. Taken together, our data suggest that AML1 haploinsufficiency leads to abnormalities in platelet that are identical to those observed in patients with FPD/AML. Thus, these mice should prove useful for exploring the molecular mechanisms through which AML regulates the normal maturation of megakaryocytes. Our early analysis suggests altered PKCδ signaling is a possible contributing factor to the observed phenotypic abnormalities.

Developmental Stage-Selective Effect of Somatically Mutated GATA-1 in Down Syndrome AML M7--a Potential Basis for Transient Myeloproliferative Disorder.

Transient myeloproliferative disorder (TMD) is a self-limited disorder of newborns with Down Syndrome (DS). Recently, acquired mutations in GATA-1 have been identified in megakaryoblasts from nearly all DS neonates with TMD, and in virtually all DS patients with the related acute megakaryoblastic leukemia (DS-AMKL, or DS-AML M7). These mutations lead to production of an N-terminus truncated GATA-1 protein (GATA-1s). Here we address the biological properties of GATA-1s. Two alternate hypotheses may account for the temporal nature of TMD and restriction of DS-AMKL to infants. First, GATA-1 somatic mutation under the DS background might occur only during a narrow developmental window and additional mutations are needed to sustain a premalignant clone; alternatively, GATA-1 mutation might occur at any time, but only fetal progenitors are sensitive to effects of GATA-1s. Our findings provide support for the latter possibility. In our study, we have generated ES cells and mice harboring a GATA-1 knockin allele with a truncation at this domain (GATA-1ΔN). ES cells bearing GATA-1ΔN exhibit slightly perturbed erythropoiesis, but generate a unique type of large thrombopoietin-dependent megakaryocytes-containing colonies, when differentiated in vitro. Remarkably, adult mutant mice have normal platelet and red blood cell counts, and their corresponding progenitors. During embryonic development, however, mutant embryos are anemic in E12.5 fetal livers, but return to normal shortly thereafter. Mutant fetal livers contain elevated numbers of CD41+ cells throughout embryonic development, especially during mid-gestation. Despite these abnormalities in proliferation, differentiation/maturation of both erythrocytes and megakaryocytes are relatively normal. Strikingly, in in vitro hematopoietic colony assays, cells from yolk sacs and early-mid stage fetal livers give rise to a large number of macroscopic hyperproliferative megakaryocytic colonies (CFU-MKs). This unique type of macroscopic CFU-MK is greatly reduced in number after mid-fetal liver stage and not detectable after birth. Interestingly, wild type mid-gestation fetal liver cells ectopically expressing GATA-1s through retroviral transduction also give rise to similar macroscopic CFU-MK colonies, as do wild type ES cells expressing GATA-1s from a GATA-1 regulatory element, when differentiated in vitro. Overall, our data suggest that the GATA-1 N-terminal domain controls proliferation of megakaryocytes, but has a more limited role in their differentiation. The N-terminal truncation of GATA-1, therefore, uncouples proliferation from differentiation, and does so dominantly. The biological effects of GATA-1s appear restricted to a unique, previously unrecognized yolk sac/fetal liver progenitor, a finding that we propose accounts for the transient nature of TMD and the infantile restriction of DS-AMKL. This observation may also pertain to other infant leukemia syndromes where the target cell may prove to be an embryonic/fetal progenitor rather than an adult myeloid progenitor. Our results thus support a model for the pathogenesis of TMD/DS-AMKL in which trisomy 21 in DS patients provides a “sensitized” background that favors exaggerated expansion of this fetal-type progenitor harboring GATA-1s. This is predicted to lead to TMD, which then evolves into full-blown DS-AMKL following additional, as yet unknown, somatic mutation(s).

The Megakaryocyte Lineage Arises in the Yolk Sac and Generates an Initial Wave of Large Embryonic Platelets in the Early Mammalian Embryo.

Two distinct waves of hematopoietic progenitors originate in the yolk sac of the mammalian embryo. The first “primitive” wave contains primitive erythroid and macrophage progenitors that generate the embryo's first red cells (Palis, et al. Development 126:5073, 1999). The second “definitive” wave consists of definitive erythroid (BFU-E) and multiple myeloid progenitors that arise later in the yolk sac and are subsequently found in the fetal liver and postnatal marrow. While megakaryocyte progenitors have been detected in the yolk sac, the ontogeny of the megakaryocyte lineage is poorly understood. Furthermore, it is not been determined when platelets first enter the bloodstream of the mouse embryo. The presence and size of platelets in the embryonic bloodstream were examined by microscopy after staining with anti-GPV antibodies. Rare platelets were identified in 2 of 4 litters of mice at E10.5. These platelets were very large with a diameter of 4.2 ± 0.4 (mean ± SEM) microns. Platelet size remained large (4.0-3.8 microns) at E11.5–E12.5, but decreased to 3.3-3.2 microns in diameter between E13.5 and E15.5 of gestation. At birth, the mean platelet diameter (2.8 ± 0.04 microns) was similar to that of adult mice (2.7 microns). These results indicate that large, embryonic platelets begin to circulate in the mouse embryo beginning at E10.5 and raise the possibility that embryonic, fetal, and adult waves of platelets are produced during mammalian embryogenesis. Using a collagen-based culture system, megakaryocyte progenitors (Meg-CFC) were first identified in late primitive streak embryos (E7.25), concomitant with primitive erythroid progenitors. Meg-CFC numbers subsequently expand in the yolk sac along with BFU-E before the development of the fetal liver. To examine the relationship of the megakaryocyte and primitive erythroid lineages in the yolk sac, we stained hematopoietic colonies grown in collagen for 5–10 days with anti-GP1bβ (megakaryocyte) and anti-βH1-globin (primitive erythroid) antibodies. As expected, the majority of the stained colonies were primitive erythroid (containing only βH1-globin-positive cells) and approximately 15% of the colonies were megakaryocyte (containing only GP1bβ-positive cells). However, 15% of the colonies contained both GP1bβ- and βH1-globin-positive cells consistent with an origin from bipotential primitive erythroid/megakaryocyte progenitors. Furthermore, proplatelet formation was evident in both unipotential and bipotential megakaryocyte colonies cultured for 10 days. Our studies support the concept that the megakaryocyte and primitive erythroid lineages originate in the yolk sac from a bipotential precursor. This parallels lineage relationships in the bone marrow which contains bipotential definitive erythroid/megakaryocyte progenitors. Finally, we hypothesize that yolk sac-derived “primitive” Meg-CFC give rise to the first embryonic platelets that enter the bloodstream soon after the onset of circulation as the fetal liver becomes a hematopoietic organ.

Comparison of Cytokine Efficacy on Ex Vivo Expansion of Megakaryocyte (MK) Progenitor Cells between Essential Thrombocythemia (ET) and Normal Person.

Essential thrombocythemia (ET) is a chronic myeloproliferative disorder (MPD) of insidious onset and characterized by regulatory defect in stem cells. It is possible that ET can induce autonomous megakaryocyte (MK) growth. The physiology of megakaryocytopoiesis was not well defined, and, as a consequence, the pathophysiology of ET characterized by excessive platelet production was poorly understood. Secondary thrombocytopenia, especially post-transplantation, could be fatal so there were many trials to overcome this problem. This experiment was designed to test the effect of cytokines on ex vivo expansion of MK progenitor cells from ET. The MK colony forming unit (CFU-MK) expansion degree according to single or combination cytokines were analyzed in normal person and ET patients. Mononuclear cells were isolated from bone marrow aspiration and then cultured in serum-free medium (MegaCult™) supplemented with SCF, G-CSF, TPO, IL-11 and 3 types of cytokine combination. After 14 days of ex vivo expansion and then the CFU-MK were counted by microscope. In the absence of cytokines, CFU-MK of ET significantly expanded than normal (p<0.05), which represented autonomous CFU-MK formation. There were significantly increased mixed CFU-MK of ET in SCF, G-CSF and TPO (SGT) combined cytokine added well (p<0.05) but pure and total CFU-MK of SGT added well did not show significant expansion. When the effect of cytokines were compared between normal and ET, all of the cytokines significantly expand CFU-MK in ET (p<0.05) than normal and especially in pure CFU-MK. In summary, autonomous MK growth was shown in ET and SGT can be relatively effective cytokine combinations in ex vivo CFU-MK expansion. Further investigation will be needed to find out the most effective culture system and cytokine combination for ex vivo MK progenitor expansion.

Synergistic effects on erythropoiesis, thrombopoiesis, and stem cell competitiveness in mice deficient in thrombopoietin and steel factor receptors

AbstractThe degree of redundancy between thrombopoietin (Tpo) and steel factor (SF) cytokine pathways in the regulation of hematopoiesis was investigated by generating mice lacking both c-Mpl and fully functional c-Kit receptors. Double-mutant c-Mpl–/–KitWv/Wvmice exhibited reduced viability, making up only 2% of the offspring from c-Mpl–/–KitWv/+intercrosses. The thrombocytopenia and megakaryocytopenia characteristic ofc-Mpl–/–mice was unchanged inc-Mpl–/–KitWv/Wvmice. However, the number of megakaryocytic colony forming units (CFU-Mks) was significantly reduced, particularly in the spleen. WhileKitWv/Wvmice, but notc-Mpl–/–mice, are anemic, the anemia was more severe in double-mutantc-Mpl–/–KitWv/Wvmice, indicating redundancy between Tpo and SF in erythropoiesis. At the primitive cell level,c-Mpl–/–andKitWv/Wvmice have similar phenotypes, including reduced progenitors, colony forming units–spleen (CFU-Ss), and repopulating activities. All of these parameters were exacerbated in double-mutant mice. c-Mpl–/–KitWv/Wvmice had 8-fold fewer clonogenic progenitor cells and at least 28-fold fewer CFU-Ss. c-Mpl–/–mice also demonstrated a reduced threshold requirement for nonmyeloablative transplant repopulation, a trait previously associated only withKitWmice, and the level of nonmyeloablative engraftment was significantly greater inc-Mpl–/–KitWv/Wvdouble mutants. Thus,c-Mpl–/–KitWv/Wvmice reveal nonredundant and synergistic effects of Tpo and SF on primitive hematopoietic cells.

Estrogen stimulates differentiation of megakaryocytes and modulates their expression of estrogen receptors alpha and beta.

Estrogen has multifunctional effects influencing growth, differentiation, and function in many tissues. High-dose estrogen has been shown to produce anabolic skeletal effects in the skeleton of postmenopausal women with increased megakaryocyte (MK) population in the bone marrow, suggesting a possible role for these cells in bone remodelling. To investigate if estrogen stimulates megakaryocytopoiesis and affects on estrogen receptor (ER) expression, CD34(+) cells were cultured for 6, 9, and 14 days plus or minus low-dose or high-dose 17 beta estradiol (E). Cells were immunolocalised for CD61, CD41, ER alpha and beta. ER mRNA expression was assessed by RT-PCR. Cells formed more CD61 positive MK colonies with low- and high-dose E treatment (P < 0.001) at 6 and 9 days. CD41 expression was increased dose-dependently in MK (3- and 5-fold P < 0.001) at 9 days. E-stimulated ER alpha expression at 6 days (P < 0.001) whilst ER beta was dose-dependently increased only at 9 days (P < 0.01). ER alpha mRNA was increased at 6 days but not at 14 days whilst ER beta mRNA expression was only increased at 14 days with E treatment. These results demonstrate that E stimulates the colony forming potential of CD34(+) cells to a more megakaryocytic phenotype in vitro. This finding together with the stimulation of ER protein and mRNA expression adds to the increasing evidence for a role for MKs in estrogen-induced bone formation.

The effect of costimulatory factors in the pathogenesis of chronic idiopathic thrombocytopenic purpura.

To investigate the effect of costimulatory factors in the pathogenesis of chronic idiopathic thrombocytopenic purpura (CITP), we examined the expression of CD80 on platelets and megakaryocytes in patients with CITP and the controls by FACS. By using CD80 monoclonal antibody (McAb) to inhibit interaction among cells which is mediated by costimulatory factors, we observed the effect of CD80 McAb on the growth and maturation of megakaryocytic progenitors of patients with CITP in vitro. The results showed the expression of CD80 on platelets and megakaryocytes in CITP group was significantly higher than that in controls (P<0.01). There was a significantly positive correlation between the expression of CD80 on platelets and serum PAIgG in CITP (r=0.86, P<0.05). The mean of various clone numbers (CFU-MK, BFU-MK and mCFU-MK) in CITP were all lower than those in controls (P<0.05). In megakaryocytes co-cultured with CD80 McAb, there was an increasing tendency of the number of CFU-MK and big CFU-MK (the number of megakaryocyte with GP IIIa positive was more than 20) and mediate CFU-MK (the number of megakaryocyte with GP IIIa positive was 11-20). When the concentration of CD80 McAb was 10 microg/L, there was a significant difference in the number of megakaryocytic colony formation (CFU-MK, BFU-MK and mCFU-MK) between the group with CD80 McAb and that without it (P<0.05). These showed the abnormality of costimulatory factors had important effect in the pathogenesis of CITP.

Characterization of Clonogenic Progenitors in Autologous Peripheral Blood Stem Cell Grafts: Evaluation of a Simple In Vitro Assay Suitable for Routine Clinical Use

Autologous peripheral blood stem cell (PBSC) transplantation has a low treatment-related morbidity and mortality when using appropriate criteria for patient selection and graft quality evaluation. It will be important to use simple and standardised procedures for evaluation of progenitor cell numbers when considering autografting in patients with malignant or non-malignant disorders and increased risk of prolonged posttransplant cytopenia. We determined the number of clonogenic cells in PBSC autografts after 7 days of In Vitro culture, and these results were compared with both the total number of colonies and the numbers of colony subsets in conventional 14 days colony assays (colony-forming unit granulocyte-erythrocyte-macrophage-megakaryocyte, CFU-GEMM; CFU-E, CFU-GM; CFU-megakaryocyte). The total colony number after 7 days of culture correlated significantly with (i) the CD34+ cell number; (ii) the total colony number as well as the numbers of erythroid, nonerythroid and mixed colonies in a conventional assay using 14 days of culture; (iii) the number of megakaryocyte colonies. The total colony number after 7 days of In Vitro culture is a simple In Vitro parameter that seems to reflect the proliferative capacity of various progenitor subsets in PBSC autografts. This simple analysis may be used in combination with other In Vitro techniques (e.g. estimation of stem cell viability and CD34+ cell subset analysis) for pretransplant evaluation of autografts. However, the possible clinical use of this parameter has to be examined in prospective clinical studies.

Effects of oxygen-induced lung damage on megakaryocytopoiesis and platelet homeostasis in a rat model.

The association between lung injury and thrombocytopenia was investigated by comparing the megakaryocyte and platelet counts, and platelet activation using P-selectin as a marker, between the prepulmonary (right atrial) and postpulmonary (left atrial) blood in adult and neonatal (preterm and term) rats with and without hyperoxic lung injury. In the healthy controls, the postpulmonary blood had lower megakaryocyte count (prepulmonary versus postpulmonary: Preterm: 8.7[0.6] versus 3.9[0.3] per ml, p < 0.001; Term: 8.7[1.1] versus 2.6[0.4] per ml, p < 0.001; Adult: median [interquartile ranges]: 2.5[1.0, 5.0] versus 1.0[0, 3.0] per ml, p < 0.001), higher platelet count (prepulmonary versus postpulmonary: Preterm: 491.2[11.1] x 10(9)/L versus 595.1[10.2] x 10(9)/L, p < 0.001; Term: 472.5[19.9] x 10(9)/L versus 579.3[26.2] x 10(9)/L, p < 0.001; Adult: 513.9[31.5] x 10(9)/L versus 664.7[28.8] x 10(9)/L, p < 0.001), but similar P-selectin expression. In contrast, the lung-damaged animals did not show any such differences in either megakaryocyte or platelet count, but P-selectin expression was greater in the postpulmonary blood (prepulmonary versus postpulmonary: Preterm: 38.7[3.9] versus 56.4[4.9]% platelets, p = 0.02; Term: 40.9[2.0] versus 54.0[4.2]% platelets, p = 0.002; Adults: 30.0[3.6] versus 49.1[4.7]% platelets, p = 0.003). Peripheral platelet and intra-pulmonary megakaryocyte counts in the lung-damaged rats were significantly lower than those in their respective controls. Intra-pulmonary thrombi or platelet aggregation were detected in the lung-damaged rats but not in the controls. These findings showed that hyperoxic lung damage reduced circulating platelets through (1) failure of the lungs to retain and fragment megakaryocytes to release platelets, and (2) platelet activation leading to platelet aggregation, thrombi formation and platelet consumption. The magnitude of platelet reduction was physiologically significant, as demonstrated by higher counts of megakaryocyte colony forming units in the bone marrow culture of the animals in the hyperoxia group when compared with the controls.

Carboxy-terminal fragment of osteogenic growth peptide in vitro increases bone marrow cell density in idiopathic myelofibrosis.

Idiopathic myelofibrosis (IMF) is a clonal stem cell disorder characterized by reactive fibrosis of bone marrow sustained by a complex cytokine network. At present, no efficacious therapy for this disease exists. Synthetic carboxy-terminal pentapeptide of osteogenic growth factor (sOGP10-14) can increase bone marrow cellularity and the number of haematopoietic colonies; this study evaluated the activity of sOGP10-14 in IMF. Fragments of bone marrow biopsies from patients affected by IMF were cultured with or without the addition of sOGP10-14. Cellular density was evaluated by image analysis, and transforming growth factor-beta1 (TGF-beta1) concentration was immunologically assayed in the supernatant of cultured bone marrow biopsies. The proliferation rate of the megakaryoblastic M07-e cell line, cultured in the presence of either granulocyte-macrophage colony stimulating factor or thrombopoietin (TPO), and with or without sOGP10-14, was evaluated. Megakaryocyte colony forming unit (CFU-Mk) assay was performed on bone marrow samples of IMF patients with or without sOGP10-14. After 14 d, bone marrow cellularity was significantly increased in samples cultured with the pentapeptide. Moreover, sOGP10-14 induced a significant increase of TGF-beta in culture supernatants. TPO-primed proliferation of M07-e was reduced by sOGP10-14, and the pentapeptide significantly reduced CFU-Mk on IMF bone-marrow-derived cells. sOGP10-14 increased ex vivo bone marrow cellularity in IMF. This action could be related to the megakaryocyte inhibition induced by the interference of this pentapeptide with growth factor activities. These findings suggest that a deficiency of osteoblast-related factors may play a role in bone marrow failure in IMF.

Thrombopoietin promotes mixed lineage and megakaryocytic colony-forming cell growth but inhibits primitive and definitive erythropoiesis in cells isolated from early murine yolk sacs

The role of thrombopoietin (Tpo) in promoting hematopoiesis has been extensively studied in late fetal, neonatal, and adult mice. However, the effects of Tpo on early yolk sac hematopoiesis have been largely unexplored. We examined whole embryos or the cells isolated from embryo proper and yolk sacs and identified both Tpo and c-mpl (Tpo receptor) mRNA transcripts in tissues as early as embryonic day 6.5 (E6.5). Presomite whole embryos and somite-staged yolk sac and embryo proper cells were plated in methylcellulose cultures and treated with selected hematopoietic growth factors in the presence or absence of Tpo. Tpo alone failed to promote colony-forming unit (CFU) formation. However, in the presence of other growth factors, Tpo caused a substantial dose-dependent reduction in primitive and definitive erythroid CFU growth in cultures containing E7.5 and E8.0 whole embryos and E8.25 to 9.5 yolk sac-derived cells. Meanwhile, Tpo treatment resulted in a substantial dose-dependent increase in CFU-mixed lineage (CFU-Mix) and CFU-megakaryocyte (CFU-Meg) formation in cultures containing cells from similar staged tissues. Addition of Tpo to cultures of sorted E9.5 yolk sac c-Kit(+)CD34(+) hematopoietic progenitors also inhibited erythroid CFU growth but augmented CFU-Mix and CFU-Meg activity. Effects of Tpo on CFU growth were blocked in the presence of a monoclonal antibody with Tpo-neutralizing activity but not with control antibody. Thus, under certain growth factor conditions, Tpo directly inhibits early yolk sac erythroid CFU growth but facilitates megakaryocyte and mixed lineage colony formation.

In VitroEffect of Interleukin-11 (IL-11) on Megakaryopoiesis from Umbilical Cord Blood Cells

Background: The megakaryopoiesis and platelet production is regulated by several hematopoietc factors such as thrombopoietin (TPO), interleukin-11 (IL-11) and interleukin-3 (IL-3). IL-11 is a potent stimulator of megakaryopoiesis in vivo, and acts primarily as a megakaryocyte maturation factor in vitro and it can act synergistically with IL-3 and TPO. We performed this study to investigate the effects of recombinant human IL-11 (rhIL-11) with other hematopoietic factors on megakaryocyte colony formation in vitro. Methods: CD34+ cells were separated from umbilical cord blood and megakaryocyte colonies using MegaCult Assay Kit were cultured with rhIL-11, recombinant human IL-3 (rhIL-3) , and recombinant human TPO (rhTPO) for 7 and 14 days. The number and percentage of CD34+ and CD41a+ cells were determined by flowcytometry. Results: The number of CD41a+ cells were 0.54±0.05×10 (rhIL-11 100 ng/ml), 5.32±0.23×10 (rhIL-3 100 ng/ml), and 8.76±0.15×10 (rhTPO 50 ng/ml) of total expanded cells during the culture of the purified CD34+ cells in liquid phase for 7 days. The number of CD41a+ cells were increased to 7.47±0.69× 10 (rhIL-3+ rhIL-11), 11.92±0.19×10 (rhTPO+rhIL-11) of total expanded cells, respectively, during the culture of the purified CD34+ cells in liquid phase for 7 days in the presence of rhIL-11 (100 ng/ml). When the purified CD34+ cells were cultured in semisolid mediaincluding various concentration of rhIL-11, the megakaryocyte colonies were not formed. When the purified CD34+ cells were cultured with rhIL-11 and rhTPO or with rhIL-11 and rhIL-3, the number of megakaryocyte colonies were increased compared with rhTPO or rhIL-3 alone. Conclusion: These results indicate that IL-11 exerts a potent proliferative activity to colony forming unit-megakaryocyte from human umbilical cord blood, and it acts with other hematopoietic factors synergistically. (Immune Network 2003;3(1):47-52)

Characterization of mouse clonogenic megakaryocyte progenitors.

Although it has been shown that unfractionated bone marrow, hematopoietic stem cells, common myeloid progenitors, and bipotent megakaryocyteerythrocyte progenitors can give rise to megakaryocyte colonies in culture, monopotent megakaryocyte-committed progenitors (MKP) have never been prospectively isolated from the bone marrow of adult mice. Here, we use a monoclonal antibody to the megakaryocyte-associated surface protein, CD9, to purify MKPs from the c-kit(+)Sca-1(-)IL7Ralpha(-)Thy1.1(-)Lin(-) fraction of adult C57BLKa-Thy1.1 bone marrow. The CD9(+) fraction contained a subset of CD41(+)FcgammaR(lo)CD34(+)CD38(+) cells that represent approximately 0.01% of the total nucleated bone marrow cells. They give rise mainly to colony-forming unit-megakaryocytes and occasionally burst-forming unit-megakaryocytes, with a plating efficiency >60% at the single-cell level. In vivo, MKPs do not have spleen colony-forming activity nor do they contribute to long-term multilineage hematopoiesis; they give rise only to platelets for approximately 3 weeks. Common myeloid progenitors and megakaryocyteerythrocyte progenitors can differentiate into MKPs after 72 h in stromal cultures, indicating that MKPs are downstream of these two progenitors. These isolatable MKPs will be very useful for further studies of megakaryopoiesis as well as the elucidation of their gene expression patterns.

Role of human interleukin-9 as a megakaryocyte potentiator in culture.

This study investigated the effect of interleukin-9 (IL-9) on the proliferation and differentiation of human colony-forming unit megakaryocytic progenitor cells (CFU-Meg). Peripheral blood-derived CD34(+)IL-6R(-) cells were sorted and cultured in the presence of IL-9, erythropoietin (Epo), stem cell factor (SCF), and thrombopoietin (TPO) alone or in combination. The number of pure and mixed megakaryocyte colonies, the size of pure megakaryocyte colonies, the ploidy distribution of megakaryocytes, and proplatelet formation were investigated. Apart from TPO, no single factor could support CFU-Meg-derived colony formation, but each two-factor combination among IL-9, Epo, and SCF supported a few CFU-Meg colonies. Interestingly, the combination of Epo+SCF+IL-9 induced four to six times as many CFU-Meg colonies as any of the two-factor combinations. Neutralizing monoclonal antibodies (mAbs) for IL-9 receptor and c-kit completely abolished this synergistic effect. In contrast, addition of neutralizing anti-c-Mpl or anti-CXCR4 Abs did not influence colony formation, indicating that this synergistic effect was independent of TPO or SDF-1. Moreover, the endogenous production of TPO by cultured CD34(+)IL-6R(-) cells in the presence of Epo+SCF+IL-9 was ruled out by reverse transcriptase polymerase chain reaction for TPO mRNA. Interestingly, the combination of TPO, Epo, SCF, and IL-9 supported the largest number of pure and mixed megakaryocyte colonies, suggesting that this combination of cytokines might recruit primitive megakaryocytic as well as multipotential progenitors. This combination also potently enhanced proplatelet formation compared with TPO alone or a combination of Epo, SCF, and IL-9. This study demonstrated for the first time that human IL-9 can potentiate human megakaryocytopoiesis in the presence of Epo and/or SCF.

Synergistic and inhibitory interactions in the in vitro control of murine megakaryocyte colony formation.

The formation of megakaryocyte colonies in agar cultures of murine bone marrow or spleen cells can be stimulated by the addition of interleukin-3 (IL-3), erythropoietin (EPO), thrombopoietin (TPO), or IL-6. However, greater numbers of colonies developed if combinations of two or more of these stimuli were used, particularly combinations including stem cell factor, with maximal numbers of colonies developing with the combination of stem cell factor plus IL-3 plus EPO. The data indicate that most committed progenitor cells in the megakaryocyte lineage were unusual in that they required stimulation by two or more hematopoietic growth factors. In tests using a range of growth factors, G-CSF was exceptional in that it consistently specifically inhibited megakaryocyte colony formation stimulated by EPO, TPO, or IL-6 but not that stimulated by IL-3. The mechanisms involved in this inhibitory action of G-CSF are unknown, but the inhibitory action could be of relevance for the dose-dependent lowering of platelet levels observed in some subjects injected with G-CSF.

Autoantibody to c-Mpl (thrombopoietin receptor) in systemic lupus erythematosus: relationship to thrombocytopenia with megakaryocytic hypoplasia.

To examine the prevalence, clinical associations, and pathogenic role of autoantibodies to c-Mpl, the thrombopoietin (TPO) receptor, in patients with systemic lupus erythematosus (SLE). Sera from 69 SLE patients, 84 patients with idiopathic thrombocytopenic purpura (ITP), and 60 healthy individuals were screened for anti-c-Mpl antibodies by enzyme-linked immunosorbent assay using recombinant c-Mpl as an antigen. Clinical findings, autoantibody profiles, and serum TPO levels were compared between SLE patients with and without anti-c-Mpl antibodies. A pathogenic role for the anti-c-Mpl antibody was evaluated by examining its inhibitory effect on TPO-dependent cell proliferation and megakaryocyte colony formation. Serum anti-c-Mpl antibody was detected in 8 SLE patients (11.6%) and 7 ITP patients (8.3%), but in none of the healthy controls. Anti-c-Mpl antibody was associated with thrombocytopenia (P = 0.0002) and a decrease in bone marrow megakaryocytes (P = 0.02) in SLE patients. Serum TPO levels in thrombocytopenic SLE patients with anti-c-Mpl antibodies were significantly elevated compared with levels in those without the antibodies (P = 0.007). IgG fractions purified from anti-c-Mpl antibody-positive sera bound to c-Mpl expressed on the cell surface and inhibited TPO-dependent cell proliferation and megakaryocyte colony formation. Autoantibody to c-Mpl is present in a subset of SLE patients with thrombocytopenia and megakaryocytic hypoplasia. It is likely that the impaired thrombopoiesis in these patients is mediated by the anti-c-Mpl antibody, which functionally blocks an interaction between TPO and c-Mpl.

In vitro megakaryocyte expansion in patients with delayed platelet engraftment after autologous stem cell transplantation

Increasing the number of megakaryocytic cells in stem cell transplants by ex vivo expansion culture may provide an approach to accelerate platelet engraftment after high-dose chemotherapy. However, it is unknown if a relationship exists between the expansion potential of progenitor cells and the time to platelet engraftment in vivo. Therefore, we questioned if those patients who potentially would benefit most from expanded cell supplements are able to generate megakaryocytic cells efficiently in vitro. The in vitro megakaryocyte proliferation was analyzed from 19 leukapheresis samples from a group of multiple myeloma patients who all showed rapid neutrophil engraftment, but varied from 7 to 115 days post-transplant to achieve platelet levels >20x10(9)/l. CD34+ cells were isolated and analyzed for their potential to form megakaryocytic colonies (CFU-Mk) in colony assays and megakaryocytic (CD61+) cells in suspension cultures. The frequency and size of CFU-Mk and the expansion potential of CD61+ cells varied eightfold between individual patients. A similar range was found with CD34+ cells isolated from normal bone marrow (n=9). Rapid platelet engraftment occurred in patients receiving both high or low CFU-Mk doses and with high and low expansion of CD61+ cells. Four patients who experienced prolonged (>3 weeks) thrombocytopenia received low CFU-Mk doses, but the expansion potential was around median values or higher. Therefore, we conclude that the megakaryocyte proliferation is not impaired and that in vitro expansion could increase the number of megakaryocytic cells, although other factors could be more relevant in platelet engraftment in this group of patients.