Megakaryocyte Ploidy Distribution Research Articles

N-Acetyl-L-Cysteine Improves Mesenchymal Stem Cell Function in Prolonged Isolated Thrombocytopenia Post-Allotransplant

Background: Prolonged isolated thrombocytopenia (PT), is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and defined as the engraftment of all peripheral blood cell lines other than a platelet (PLT) count ≤20×10E+9/L or dependence on PLT transfusions for more than 60 days after allo-HSCT. Several clinical risk factors have been proposed to be associated with PT after allo-HSCT. However, the underlying mechanisms remain to be elucidated. The processes of megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, cytokines, and bone marrow (BM) stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. However, little is known about the quantity and function of BM MSCs and how to improve the impaired BM MSCs in PT patients.AimsTo evaluated the number and function of BM MSCs in patients with PT post-allotransplant. Moreover, to investigate the approach to enhance the number and function of BM MSCs derived from patients with PT and its underlying molecular mechanisms in vitro.MethodsThree cohorts were included: patients with PT (N=83), patients with good graft function (GGF, N=32), defined as persistent successful engraftment after allotransplant, and transplant donors as normal controls (N=15). The number and functions of BM MSCs were evaluated by fibroblasts colony-forming unit (CFU-F) assay, cell proliferation assay and senescence-associated β-galactosidase (SAβ-gal) assay. Reactive oxygen species (ROS) levels and protein expression for p-p38, p38, p-p53, p53 was measured by flow cytometry. The number of megakaryocyte (MK), MK apoptosis, MK ploidy distribution and platelet release values after coculture were assessed by flow cytometry. To further investigate the potential effect for repairing the dysfunctional BM MSCs, N-Acetyl-L-cysteine (NAC,a ROS scavenger), SB203580 (p38 inhibitor) and PFT-a (p53 inhibitor) were administrated to the BM MSCs from PT patients. After 2 days in vitro culture, the percentage of SAβ-positive cells, the intracellular levels of ROS, p-p38, p-p53, the number and the function of MK were evaluated. For evaluation of the efficacy and safety of NAC in PT patients, 20 patients with PT were subsequently enrolled to a single-center pilot study.ResultsHuman BM MSCs were demonstrated as spindle shape and typical immunophenotype of MSCs at day 21 of cultivation among subjects with PT, GGF and normal controls. 7 of the 32 GGF BM and 58 of the 83 PT BM failed to produce any adherent layers within 3 weeks of culture. BM MSCs derived from PT patients expanded more slowly and appeared flattened and larger. Proliferative capacity and CFU-F counts of BM MSCs from PT patients were significantly reduced compared to those of GGF patients and normal controls. Moreover, increased levels of ROS, which was associated with increased number of SAβ-positive cells, were identified in BM MSCs from PT patients. Intracellular p-p38 and p-p53 levels were significantly elevated in PT patients compared to those in GGF patients. Moreover, the percentages of MK were lower in CD34+ cells cocultured with PT-MSCs compared to those observed with GGF-MSCs. CFU-MKs counts were also lower in the PT-MSCs group. After NAC treatment in vitro, the proliferative capacity was increased significantly, whereas the number of senescent cells, the intracellular levels of ROS, p-p38 and p-p53 were reduced markedly in BM MSCs from PT patients. NAC exposure also improved the ability of BM MSCs from PT patients to support megakaryocytopoiesis. The preliminary clinical data of a single-center pilot study showed the complete response, response, and overall response rates at 2 months after NAC treatment (400 mg three times a day) were 10% (2/20), 55% (11/20) and 65% (13/20), respectively, and no apparent drug-associated side effects were observed.Summary/ConclusionIn summary, the current study demonstrated the abnormalities of BM MSCs contribute to the pathogenesis of PT following allo-HSCT. Moreover, BM MSCs derived from PT patients exhibited quantitative and functional improvement following in vitro treatment with NAC. Although these findings require further validation, our in vitro data and preliminary clinical data suggested NAC is an effective and safe treatment option for PT patients after allo-HSCT. DisclosuresNo relevant conflicts of interest to declare.

Characterization of thrombopoietin kinetics within 60 days after allogeneic hematopoietic stem cell transplantation and its correlation with megakaryocyte ploidy distribution.

Thrombopoietin (TPO) has been identified as a key cytokine for both megakaryogenesis and thrombopoiesis. We attempt to characterize the kinetics of TPO and its correlation with megakaryocytes (MKs) ploidy distribution pattern within 60 d after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Forty-six consecutive patients undergoing allo-HSCT from October 2008 to December 2008 were included. TPO levels and ploidy distribution patterns of MKs were measured using ELISA and flow cytometric analysis, respectively. The results indicated that TPO levels and the platelet count followed opposite trends after allo-HSCT. The preconditioning TPO levels and the number of transplanted CD34(+) cells were significant predisposing factors for rapid platelet engraftment (p = 0.010 and 0.007, respectively) by multivariate analysis. There was a reduction of ploidy and an increase in immature MKs in patients with higher endogenous TPO levels (>250 pg/mL) on day 60 after allo-HSCT. Moreover, lower TPO levels (≤250 pg/mL) on day 60 after allo-HSCT were associated with significantly improved five-yr overall survival (p = 0.021) and reduced transplant-related mortality (p = 0.033). In conclusion, endogenous TPO levels may be associated with platelet recovery and have prognostic significance during allo-HSCT.

Characterization of TPO Kinetics within 60 Days of Allogeneic Hematopoietic Stem Cell Transplantation and Its Correlation with Megakaryocyte Ploidy Distribution

Abstract 4463 Background:Thrombocytopenia is a critical complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), but its pathogenesis has remained obscure. Thrombopoietin (TPO) has been identified as a key cytokine for both megakaryogenesis and thrombopoiesis; however, the kinetics of TPO production after allo-HSCT has not been reported. This study characterized the kinetics of TPO and its correlation with the megakaryocyte ploidy distribution pattern within 60 days of allo-HSCT. Methods and Results:A total of 46 consecutive patients who underwent allo-HSCT from October 2008 to December 2008 were included in the study. The TPO levels and the ploidy distribution pattern of MKs were measured using ELISA and flow cytometric analysis, respectively. The results indicate that the TPO levels and the platelet counts followed opposite trends after allo-HSCT. Multivariate analysis indicate that endogenous TPO levels before allo-HSCT, and the number of transplanted CD34+ cells were significant predisposing factors for rapid platelet engraftment (P=0.010 and 0.007, respectively). Furthermore, we found a reduction of ploidy and an increase in immature MKs in patients with higher endogenous TPO levels (> 250 pg/ml) on day 60 after allo-HSCT. Moreover, lower TPO levels (≤250 pg/ml) on day 60 after allo-HSCT were associated with significantly improved 2-year OS (P=0.032) and reduced TRM (P=0.026). Conclusion:Our results suggest that increased endogenous TPO levels may not be sufficient after allo-HSCT, and that the decreased ability of TPO may account for the appearance of antibodies. The use of exogenous rhTPO may promote platelet recovery after allo-HSCT. However, these possibilities need to be examined further. Disclosures:No relevant conflicts of interest to declare.

RhoA Is Essential for Maintaining Normal Megakaryocyte Ploidy Distribution and Platelet Generation

Abstract 385The small GTPase, RhoA orchestrates actin cytoskeletal dynamics, which plays a role in platelet development and function. After platelet activation, RhoA rearranges the cytoskeleton by facilitating shape change, granule release, and clot retraction. In addition, RhoA is involved in platelet development. It does this by presumably regulating cytokinesis during megakaryocyte-erythroid progenitor cell expansion and megakaryopoiesis. RhoA also regulates thrombopoiesis by coordinating end bifurcation of the proplatelet extensions to amplify preplatelet numbers and the regulation of platelet release. Previously, most studies utilized pharmacological Rho inhibitors, such as C3 exotoxin. This could potentially obfuscate the data because of the incomplete knockdown of RhoA, the non-specific disruption of closely related Rho family members, or the long incubation times that could alter platelet biology. Therefore, we developed a transgenic mouse model that knocked out RhoA only in megakaryocytes and in platelets by using a CRE-LOX strategy to further investigate the role of RhoA in platelet biology. First, mice were generated that had loxP sites flanking the 3rd exon of RhoA (RhoAfl/fl). These mice were then crossed with mice expressing CRE recombinase driven by the platelet factor 4 promoter (PF4 CRE+), thus limiting CRE expression only in megakaryocytes and in platelets. The offspring, RhoAfl/fl PF4CRE+ mice were phenotypically normal, and had normal complete blood counts, except for macrothrombocytopenia. Their platelet counts were 25 ±3% of that observed in their littermate controls, (RhoAfl/fl PF4CRE−) and their platelet size was 130 ±10% of their littermate controls. The lack of RhoA only disrupted aggregation, granule release, and clot retraction when stimulated at the lowest dosage of agonists. To determine the causes of macrothrombocytopenia in RhoAfl/fl PF4CRE+ mice, histological examination of the spleen showed that 26.0±13.3% of the megakaryocytes had pyknotic nuclei as compared to 1.0±0.5% of the controls. In the bone marrow, apoptosis was present in 6.8±3.3% of the RhoA null megakaryocytes, but only in 1.2 ±0.3% of the control megakaryocytes. Furthermore, flow cytometry revealed that megakaryocyte counts in the bone marrow were 51.5 ±4.2% lower than in that of the controls. To determine if lacking RhoA impairs normal megakaryocyte maturation, we measured DNA ploidy using propidium iodide and flow cytometry. In megakaryocytes derived from adult bone marrow or from cultured fetal livers (E13.5, 8 days culture), the RhoAfl/fl PF4CRE+ cells had higher ploidy, a lower number of 2N cells, and an increased number of 16N cells than megakaryocytes derived from control animals. Together these data show that loss of RhoA causes deficiency of megakaryocytes probably due to increased apoptosis, and also causes aberrant maturation of the surviving megakaryocyte. To analyze whether RhoA was also required for thrombopoiesis, cultured RhoA-null megakaryocytes derived from fetal livers were infused into recipient mice. The megakaryocytes lacking RhoA more rapidly release platelets during the first 3 hours post infusion than controls. However, unlike control platelets, the Rho-null platelets were essentially gone within 24 hours. We analyzed whether the increased release of knockout platelets could be due to the up-regulation of proplatelet generation since the deletion of RhoA might impair myosin activity and impair cortical tension. However, micropipette aspiration analysis, which mimics the shear forces found in the bone marrow sinusoid capillaries, showed that the RhoA-null megakaryocytes were less compliant than the controls, primarily due to their large size. These data suggest that the higher ploidy and larger sizes of RhoA knockout megakaryocytes causes them to lodge in the pulmonary capillary bed more quickly, and to rapidly release defective macrothrombocytes. In contrast, the smaller (and thereby more compliant) wild type-cell megakaryocytes fragmented more slowly into platelets through proplatelet extension. Together, our findings demonstrate that RhoA is essential for normal megakaryocyte survival, maturation, and thrombopoiesis. Disclosures:No relevant conflicts of interest to declare.

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.

Effects of the combination of thrombopoietin with cytokines on the survival of X-irradiated CD34(+) megakaryocytic progenitor cells from normal human peripheral blood.

Combinations of thrombopoietin and cytokines that act on megakaryocyte development (stem cell factor, IL3, IL6, IL11, flt3 ligand (now known as FLT3LG), erythropoietin, GM-CSF and G-CSF were evaluated for their ability to enhance clonal growth in vitro of X-irradiated CD34(+) megakaryocytic progenitor cells (CFU-megakaryocytes) purified from normal human peripheral blood. These data were compared with corresponding results described previously for CD34(+) CFU-megakaryocytes from human placental/umbilical cord blood (I. Kashiwakura, Radiat. Res. 153, 144-152, 2000). All cytokines, except IL3, promoted thrombopoietin-induced colony formation, but they resulted in exponential radiation survival curves. No significant differences in the D(0) (46-61 cGy) and extrapolation number n (1.00-1.04) were observed between thrombopoietin alone and in combination with these cytokines. IL3 did not promote colony formation, but marked shoulders were observed on the survival curves (D(0) = 91 cGy, n = 2.83). Flow cytometric analysis of cells harvested from cultures of X-irradiated cells stimulated with thrombopoietin plus IL3 showed no significant differences in the expression of surface antigens and DNA ploidy distribution of megakaryocytes from the control. These findings suggest that IL3 plays a key role in promoting the survival of X-irradiated CD34(+) CFU-megakaryocytes from peripheral blood as well as those from cord blood, though the former are more radiosensitive.

Effects of glycosaminoglycans on the in vitro colony formation of CD34+ megakaryocytic progenitor cells in human placental/umbilical cord blood

The in vitro effect of various glycosaminoglycans (GAGs) on the clonal growth of CD34+ megakaryocytic progenitor cells (CFU-Megs) isolated from human placental/umbilical cord blood (CB) was evaluated in human plasma containing semisolid culture stimulated by recombinant human thrombopoietin (TPO). The GAGs, including hyaluronic acid from human umbilical cords (HA-h), pig skins (HA-p) and rooster combs (HA-r), or keratan sulfate (KS), various chondroitin sulfates (CS-A, B, C, D, E), and heparan sulfate (HS), were tested. Each GAG alone did not affect the clonal growth of CFU-Meg. In the presence of TPO, adding of HA-p or HS (100 micrograms/ml) resulted in an approximately 1.3-fold increase, in the total number of colonies, due to an increase in large megakaryocyte colonies. In contrast, CS-E led to a marked decrease in CFU-Meg growth. At the end of the culture, the total number of cells increased 3.0-fold of the initial value of the control, but adding HA-p or HS showed an approximately 9.1-fold or 18.3-fold increase. Similarly, the total number of CFU-Meg detected in the harvested cells increased to 4.8-fold of the initial value, while, an approximately 18.3-fold or 38.8-fold increase was observed in the culture containing HA-p or HS, respectively. Flow cytometric analysis of the harvested cells showed no significant difference in the expression of surface antigens and DNA ploidy distribution of megakaryocytes between the control and GAG treatments. These results suggest that HA-p and HS promote the proliferation of immature CB CD34+ CFU-Meg in the presence of TPO.

Megakaryocyte ploidy and platelet changes in human diabetes and atherosclerosis.

Altered platelet morphology and function have been reported in patients with diabetes. They are likely to be associated with the pathological processes and increased risk of vascular disease seen in these patients. Mean platelet volume (MPV), platelet count, and megakaryocyte (MK) ploidy (DNA content) were measured in (1) nondiabetics with normal coronary arteries, (2) nondiabetics with coronary artery atherosclerosis, (3) diabetics without evidence of vascular complications, and (4) diabetics with vascular disease. The platelet count (+/- SD) was increased in all groups but only significantly in the diabetics with vascular disease (236 +/- 65 versus 250 +/- 54 versus 257 +/- 64 versus 295 +/- 90 [P < or = .05] x 10(9)/L, for groups, I, II, II, and IV, respectively). The MPV was significantly increased in patients with atherosclerosis (7.0 +/- 0.4 versus 8.0 +/- 1.2 [P < or = .05] versus 7.2 +/- 0.9 versus 8.1 +/- 0.9 [P < or = .05] IL). Geometric mean MK ploidy was significantly increased in all groups compared with controls (16 +/- 1.5 versus 18.7 +/- 1.8 [P < or = .05] versus 19.8 +/- 1.6 [P < or = .05] versus 20.1 +/- 2.7 [P < or = .05]). Furthermore, some patients with vascular disease and/or diabetes had a modal ploidy shift from 16 (the normal mammalian modal ploidy) to 32, with a concomitant reduction of MKs in the 8 and 16 ploidy classes. This shift was seen particularly in the diabetics with vascular disease (P = .007). Interleukin-6 (IL-6) levels were measured and were elevated in patients with atherosclerosis; the highest levels were found in the diabetic patients (0.7 +/- 0.9 versus 5.3 +/- 5.5 [P < or = .05] versus 2.5 +/- 2.8 versus 6.7 +/- 5.5 [P < or = .05] ng/L). In the diabetic patients with atherosclerosis, fibrinogen levels were also increased (2.85 +/- 0.76 versus 3.34 +/- 1.32 versus 2.43 +/- 1.50 versus 5.59 +/- 1.72 [P < or = .05] g/L). Furthermore, IL-6 levels correlated with MK ploidy (r = .45, P = .009) and fibrinogen levels (r = .5, P = .0001). This study demonstrates that patients with vascular disease, particularly diabetics, have an altered MK ploidy distribution, showing a shift toward higher ploidy in association with an increased platelet mass (count x volume). Changes in platelets in diabetes probably reflect MK changes, which themselves are a response to systemic change.

6 Megakaryocytes and platelets in vascular disease

Platelets are anucleate cells with no DNA. They are derived from their precursor, the megakaryocyte (MK), whose differentiation is characterized by nuclear polyploidization through a process called endomitosis. Changes in the MK-platelet-haemostasis axis may precede acute thrombotic events. Changes in MK ploidy distribution may be associated with the production of large platelets. Mean platelet volume (MPV) is an important biological variable as it is a determinant of platelet reactivity. Large platelets are denser and more active haemostatically. MPV is increased in patients after myocardial infarction (MI) and is a predictor of a further ischaemic event and death when measured after MI. It has been suggested that changes not only in platelets but also in the parental MK are associated with chronic and acute vascular events. The regulation of megakaryocytopoiesis depends on several haematopoietic factors such as thrombopoietin. An understanding of the signalling system that controls platelet number and size might give insight into a role of platelet production in thrombogenesis and atherogenesis.

Evaluation of ploidy of mature canine megakaryocytes, using Feulgen staining and microspectrophotometry

Abstract Objective To evaluate megakaryocyte size and ploidy, using Feulgen staining and microspectrophotometry, in adult dogs with normal platelet count. Animals Group A contained 8 and group B contained 11 adult dogs. Procedure Megakaryocytes were evaluated by light microscopy and staged according to maturation status. Stage-III megakaryocytes were measured and mapped for future relocation. Bone marrow aspirates were destained and restained, using the Feulgen method. Previously identified stage-III megakaryocytes were measured for DNA content, using microspectrophotometry. Results Megakaryocyte size correlated with ploidy values, and mean sizes within ploidy groups were significantly (P &lt; 0.05) different from each other for both groups. The modal ploidy value of stage-III megakaryocytes, which represented 18% of the total megakaryocyte population of the combined groups, was 32N. This is in contrast to results of flow cytometric studies, which indicated that the modal ploidy value for all canine megakaryocytes was 16N. Conclusions Reasons for the disparate results between microspectrophotometric techniques and flow cytometry include maturation stage of the megakaryocyte population evaluated and percentage of megakaryocytes within that maturation stage. Flow cytometric methods, which evaluate all megakaryocytes detectable by antibody, may include cells still capable of DNA synthesis, resulting in a shift in the observed modal ploidy value. Recognition of the difference between canine and human megakaryocyte ploidy distribution is important, particularly in studies in which the dog is used as an animal model for human megakaryocytopoiesis. (Am J Vet Res 1996;57:1434–1437)

A Mathematical Model, Describing the Kinetic Behaviour of the Thrombopoietic System in Mice and Rats

A mathematical model of thrombopoiesis in small rodents, principally rats, is presented. It is a compartmental model, consisting of seven compartments, which contain the following cell populations: early megakaryocyte progenitor cells, late megakaryocyte progenitor cells, megakaryocytes of ploidy classes 8N, 16N, 32N and 64N, and platelets. Signals for feedback regulation are derived from the platelet compartment and from all megakaryocyte compartments. The main characteristic of the model is its ability to simulate megakaryocyte ploidy and volume distribution. Platelet number and the ploidy distribution of megakaryocytes after acute and chronic disturbances in the platelet compartment were simulated and were found to approximate experimental data published in the literature. The kinetic parameters for the progenitor cell compartment, which were derived from published in vitro data, were sufficient to stimulate the platelet number after administration of hydroxyurea and 5-fluorouracil. The proposed model is capable of reproducing the reactions of the system to several experimental disturbances in the thromobopoietic lineage, but the action of agents such as radiation, which cause damage to the stem cells or to other lineages, cannot be simulated.

Size and ploidy of rabbit megakaryocytes in dietary hypercholesterolaemia

The number and sizes of blood platelets, the blood volume, and the sizes and ploidies of megakaryocytes were measured in New Zealand white rabbits that were hypercholesterolaemic as a result of a high cholesterol diet (0.5% cholesterol + 2% peanut oil) that was continued for as long as 19 weeks. Mean platelet volumes were lower than normal, but platelet counts, platelet mass/μl of blood, and blood volumes were normal. The ploidy distribution of megakaryocytes was normal, with 32N being the predominant ploidy group in both hyper- and normocholesterolaemic animals. The average sizes of all mature megakaryocytes and of those within the 16N ploidy group were less than normal. These findings suggest that hyperlipidaemia somehow limits the expansion of megakaryocyte cytoplasm and causes it to fragment into platelets of smaller than normal size.

Stimulation of megakaryocytopoiesis in mice by human recombinant interleukin-6

The in vivo effects of purified human recombinant interleukin-6 (IL-6) on murine megakaryocytopoiesis were examined. IL-6 was administered subcutaneously to Swiss Webster mice, followed by evaluation of bone marrow megakaryocyte ploidy, size and frequency, and median platelet volume 24, 48, and 72 hours after the initiation of IL-6 administration. In addition, bone marrow megakaryocyte morphology was examined using electron microscopy at 72 hours. IL-6 (10,000 U per subcutaneous injection) was administered three times during the first 24 hours, three times during the second 24 hours, and twice during the last 24-hour period. IL-6 bioactivity (10 U/ng) was determined using the IL-6-dependent murine hybridoma cell line B9. Megakaryocyte ploidy distribution, measured by two-color flow cytometry, demonstrated a shift in the modal ploidy class from 16N to 32N and a significant increase in the relative frequency of 64N megakaryocytes 48 and 72 hours (but not 24 hours) after initiation of IL-6 administration (cumulative doses of 60,000 and 80,000 U at 48 and 72 hours, respectively). In addition, ploidy levels were increased in animals that received a cumulative IL-6 dose of only 40,000 U (evaluated after 72 hours). The size of recognizable bone marrow megakaryocytes, determined by the cross-sectional areas of plastic embedded bone marrow megakaryocytes, was increased at the 48-hour (60,000 U IL-6) and 72- hour (80,000 U IL-6) time points. Megakaryocyte frequency, measured by flow cytometry, was unaffected at all time points and doses of IL-6. Median platelet volume, measured by electrical impedance, was not consistently altered by administration of IL-6. Electron microscopic examination of bone marrow megakaryocytes showed an increase in the proportion of megakaryocytes with a wide, peripheral, organelle- deficient zone from 20% +/- 9% (SD) in control animals to 50% +/- 7% (SD) (P less than .02) in animals that received IL-6. No changes were observed in the distribution of the demarcation membranes. IL-6 is a potent stimulator of murine megakaryocytopoiesis, in vivo, and appears to act early in megakaryocyte differentiation.

Stimulation of Megakaryocytopoiesis in Mice by Human Recombinant Interleukin-6

The in vivo effects of purified human recombinant interleukin-6 (IL-6) on murine megakaryocytopoiesis were examined. IL-6 was administered subcutaneously to Swiss Webster mice, followed by evaluation of bone marrow megakaryocyte ploidy, size and frequency, and median platelet volume 24, 48, and 72 hours after the initiation of IL-6 administration. In addition, bone marrow megakaryocyte morphology was examined using electron microscopy at 72 hours. IL-6 (10,000 U per subcutaneous injection) was administered three times during the first 24 hours, three times during the second 24 hours, and twice during the last 24-hour period. IL-6 bioactivity (10 U/ng) was determined using the IL-6-dependent murine hybridoma cell line B9. Megakaryocyte ploidy distribution, measured by two-color flow cytometry, demonstrated a shift in the modal ploidy class from 16N to 32N and a significant increase in the relative frequency of 64N megakaryocytes 48 and 72 hours (but not 24 hours) after initiation of IL-6 administration (cumulative doses of 60,000 and 80,000 U at 48 and 72 hours, respectively). In addition, ploidy levels were increased in animals that received a cumulative IL-6 dose of only 40,000 U (evaluated after 72 hours). The size of recognizable bone marrow megakaryocytes, determined by the cross-sectional areas of plastic embedded bone marrow megakaryocytes, was increased at the 48-hour (60,000 U IL-6) and 72-hour (80,000 U IL-6) time points. Megakaryocyte frequency, measured by flow cytometry, was unaffected at all time points and doses of IL-6. Median platelet volume, measured by electrical impedance, was not consistently altered by administration of IL-6. Electron microscopic examination of bone marrow megakaryocytes showed an increase in the proportion of megakaryocytes with a wide, peripheral, organelle-deficient zone from 20% +/- 9% (SD) in control animals to 50% +/- 7% (SD) (P less than .02) in animals that received IL-6. No changes were observed in the distribution of the demarcation membranes. IL-6 is a potent stimulator of murine megakaryocytopoiesis, in vivo, and appears to act early in megakaryocyte differentiation.

Megakaryocyte ploidy distribution by flow cytometry

Megakaryocyte ploidy distribution by flow cytometry

Regulation of Thrombopoiesis: Effects of the Degree of Thrombocytopenia on Megakaryocyte Ploidy and Platelet Volume

AbstractWe have established a murine model and techniques with which to serially study thrombocytopoiesis after induction of experimental immune thrombocytopenia of variable severity and duration. Bone marrow megakaryocyte ploidy distribution was determined by using unfractionated bone marrow, a polyclonal megakaryocyte-specific probe, and two-color, fluorescence-activated flow cytometry. With these techniques, the modal megakaryocyte ploidy class in normal murine bone marrow was 16N. Serial studies of bone marrow megakaryocyte ploidy after the induction of acute, severe thrombocytopenia (platelet count, <0.05 x 106 μL) demonstrated no detectable change in the ploidy distribution at 12, 24, and 36 hours after the onset of thrombocytopenia. At 48 hours, the modal ploidy class shifted from 16N to 32N, and the 64N class increased significantly (P < .001). The ploidy distribution returned to normal 120 hours after the onset of thrombocytopenia. A lesser degree of thrombocytopenia (platelet count reduction to 0.100 to 0.200 x 106/μl) delayed the modal ploidy class shift from 16N to 32N until 72 hours after the onset of thrombocytopenia. Chronic, severe thrombocytopenia (platelet count, <0.05 x 106/μL for seven days) resulted in a modal ploidy class shift from 16N to 32N during the thrombocytopenic phase and an enhanced increase in the 64N megakaryocyte class during the recovery phase. Mean platelet volume (MPV) was simultaneously measured on isolated total platelet populations after induction of thrombocytopenia. MPV was significantly increased (P < .001) as early as eight hours after the onset of acute, severe thrombocytopenia, 40 hours before a shift in the ploidy distribution. Mild thrombocytopenia (platelet count reduction to 0.400 x 106/μL) was not associated with a ploidy shift but did result in a significantly increased MPV (P < .001). These studies demonstrate that the temporal relationship and magnitude of the effects of thrombocytopenia upon megakaryocyte ploidy distribution are dependent upon the degree and the duration of the thrombocytopenic stimulus and that the effects of experimental thrombocytopenia on platelet volume and megakaryocyte ploidy are dissociated. © 1987 by Grune & Stratton, Inc.

Regulation of thrombopoiesis: effects of the degree of thrombocytopenia on megakaryocyte ploidy and platelet volume

We have established a murine model and techniques with which to serially study thrombocytopoiesis after induction of experimental immune thrombocytopenia of variable severity and duration. Bone marrow megakaryocyte ploidy distribution was determined by using unfractionated bone marrow, a polyclonal megakaryocyte-specific probe, and two-color, fluorescence-activated flow cytometry. With these techniques, the modal megakaryocyte ploidy class in normal murine bone marrow was 16N. Serial studies of bone marrow megakaryocyte ploidy after the induction of acute, severe thrombocytopenia (platelet count, less than 0.05 X 10(6) microL) demonstrated no detectable change in the ploidy distribution at 12, 24, and 36 hours after the onset of thrombocytopenia. At 48 hours, the modal ploidy class shifted from 16N to 32N, and the 64N class increased significantly (P less than .001). The ploidy distribution returned to normal 120 hours after the onset of thrombocytopenia. A lesser degree of thrombocytopenia (platelet count reduction to 0.100 to 0.200 X 10(6)/microL) delayed the modal ploidy class shift from 16N to 32N until 72 hours after the onset of thrombocytopenia. Chronic, severe thrombocytopenia (platelet count, less than 0.05 X 10(6)/microL for seven days) resulted in a modal ploidy class shift from 16N to 32N during the thrombocytopenic phase and an enhanced increase in the 64N megakaryocyte class during the recovery phase. Mean platelet volume (MPV) was simultaneously measured on isolated total platelet populations after induction of thrombocytopenia. MPV was significantly increased (P less than .001) as early as eight hours after the onset of acute, severe thrombocytopenia, 40 hours before a shift in the ploidy distribution. Mild thrombocytopenia (platelet count reduction to 0.400 X 10(6)/microL) was not associated with a ploidy shift but did result in a significantly increased MPV (P less than .001). These studies demonstrate that the temporal relationship and magnitude of the effects of thrombocytopenia upon megakaryocyte ploidy distribution are dependent upon the degree and the duration of the thrombocytopenic stimulus and that the effects of experimental thrombocytopenia on platelet volume and megakaryocyte ploidy are dissociated.

Two-Color Flow Cytometric Measurement of DNA Distributions of Rat Megakaryocytes in Unfixed, Unfractionated Marrow Cell Suspensions

AbstractThe ploidy distribution of megakaryocytes shifts in response to platelet demand and thus provides a sensitive index of megakaryocytopoiesis. Flow cytometry (FCM) is a potentially valuable method for rapid determination of ploidy distributions of megakaryocyte populations; however, because megakaryocytes constitute only a very small proportion of the cells in unfractionated marrow, other rare events, such as cell clumping, complicate FCM analysis. We describe the measurement of cellular DNA distributions of megakaryocytes by two-color FCM in unfixed, unfractionated marrow—a method based on the resistance of megakaryocytes to hypotonic lysis in the cold for at least 2 days. Specific platelet antiserum was used to label megakaryocytes by indirect immunofluorescence with fluorescein (green fluorescence), and DNA was stained with propidium iodide (red fluorescence) in hypotonic citrate solution. The ploidy distribution of megakaryocytes was selectively determined with two-color, green-gated FCM, with which the red and green fluorescence of all cells is analyzed, but only the red fluorescence (DNA content) of cells that specifically bound the platelet antibody is recorded. We demonstrate that this method can readily detect changes in megakaryocyte DNA distributions due to experimental thrombocytopenia or platelet hypertransfusion and, therefore, should be useful for both experimental and clinical investigations of megakaryocytopoiesis.

Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions

The ploidy distribution of megakaryocytes shifts in response to platelet demand and thus provides a sensitive index of megakaryocytopoiesis. Flow cytometry (FCM) is a potentially valuable method for rapid determination of ploidy distributions of megakaryocyte populations; however, because megakaryocytes constitute only a very small proportion of the cells in unfractionated marrow, other rare events, such as cell clumping, complicate FCM analysis. We describe the measurement of cellular DNA distributions of megakaryocytes by two- color FCM in unfixed, unfractionated marrow--a method based on the resistance of megakaryocytes to hypotonic lysis in the cold for at least 2 days. Specific platelet antiserum was used to label megakaryocytes by indirect immunofluorescence with fluorescein (green fluorescence), and DNA was stained with propidium iodide (red fluorescence) in hypotonic citrate solution. The ploidy distribution of megakaryocytes was selectively determined with two-color, green-gated FCM, with which the red and green fluorescence of all cells is analyzed, but only the red fluorescence (DNA content) of cells that specifically bound the platelet antibody is recorded. We demonstrate that this method can readily detect changes in megakaryocyte DNA distributions due to experimental thrombocytopenia or platelet hypertransfusion and, therefore, should be useful for both experimental and clinical investigations of megakaryocytopoiesis.

Effects of endotoxin treatment on the differentiation of guinea pig megakaryocytes to blood platelets.

The effect of endotoxin treatment (30 micrograms/kg body weight) on megakaryocyte development and platelet production in guinea pigs was investigated. A moderate thrombocytopenia was noted at 24 h after endotoxin administration. Recovery was observed at 48 h when the platelet count had almost reached the normal level. A significant increase in platelet production was obtained at 48 h, indicated by an enhancement in incorporation of labelled sulphate into platelets and also by an increase in the number of circulating heavier platelets. Megakaryocytes were classified into different developmental stages - megakaryoblast, promegakaryocyte, megakaryocyte - using standard morphological criteria (size, shape of nucleus, nucleus/cytoplasm ratio). The number of mature megakaryocytes increased at 24 and 48 h after endotoxin treatment. It was possible to distinguish between two different types of mature megakaryocytes - early mature and mature megakaryocytes - by the presence of a methanol-sensitive acid phosphatase. This enzyme is found mainly in mature, presumably platelet-forming, megakaryocytes and in platelets. The increased number of mature megakaryocytes at 24 h was due to an enhancement in the number of early mature megakaryocytes, whereas the change at 48 h was due largely to an increased number of mature, platelet-forming megakaryocytes. Megakaryocytes were isolated from bone marrow of guinea pigs by Percoll density-gradient centrifugation and incubated with radioactively labelled leucine. Compared with the untreated controls megakaryocytes isolated 24 h after endotoxin treatment took up and incorporated higher amounts of leucine into protein, indicating a higher metabolic capacity of the early mature megakaryocytes. No alteration in the ploidy distribution of megakaryocytes was noted after endotoxin treatment.