Megakaryocyte Growth Research Articles

GATA1 in Normal and Pathologic Megakaryopoiesis and Platelet Development.

GATA1 is a highly conserved hematopoietic transcription factor (TF), essential for normal erythropoiesis and megakaryopoiesis, that encodes a full-length, predominant isoform and an amino (N)terminus-truncated isoform GATA1s. It is consistently expressed throughout megakaryocyte development and interacts with its target genes either independently or in association with binding partners such as FOG1(friend of GATA1). While the N-terminus and zinc finger have classically been demonstrated to be necessary for the normal regulation of platelet-specific genes, murine models, cell-line studies, and human case reports indicate that the carboxy-terminal activation domain and zinc finger also play key roles in precisely controlling megakaryocyte growth, proliferation, and maturation. Murine models have shown that disruptions to GATA1 increase the proliferation of immature megakaryocytes with abnormal architecture and impaired terminal differentiation into platelets. In humans, germline GATA1 mutations result in variable cytopenias, including macrothrombocytopenia with abnormal platelet aggregation and excessive bleeding tendencies, while acquired GATA1s mutations in individuals with trisomy 21 (T21) result in transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS) arising from a megakaryocyte-erythroid progenitor(MEP). Taken together, GATA1 plays a key role in regulating megakaryocyte differentiation, maturation, and proliferative capacity. As sequencing and proteomic technologies expand, additional GATA1 mutations and regulatory mechanisms contributing to human diseases of megakaryocytes and platelets are likely to be revealed.

Regulation of megakaryo/thrombopoiesis by endosomal toll-like receptor 7 and 8 activation of CD34+ cells in a viral infection model

BackgroundCD34+ cells, megakaryocytes (MKs), and platelets express toll-like receptors (TLRs) that enable these cells to amplify the host innate immune response. However, the role of TLR7/TLR8 activation in megakaryopoiesis has not yet been investigated. ObjectivesWe evaluated the effect of coxsackievirus B3 (CVB3) and synthetic TLR7/TLR8 agonists on the development of human MKs and production of platelets. MethodsCD34+ cells from human umbilical cord were inoculated with CVB3 or stimulated with synthetic TLR7/TLR8 agonists and then cultured in the presence of thrombopoietin. ResultsCD34+ cells, MK progenitor cells, and mature MKs expressed TLR7 and TLR8, and exposure to CVB3 resulted in productive infection, as determined by the presence of viral infectious particles in culture supernatants. Cell expansion, differentiation into MKs, MK maturation, and platelet biogenesis were significantly reduced in CD34+-infected cultures. The reduction in MK growth was not due to an alteration in cellular proliferation but was accompanied by an increase in cellular apoptosis and pyroptosis. Impairment of MK generation and maturation of viable cells were also associated with decreased expression of transcription factors involved in these processes. These effects were completely abrogated by TLR7 but not TLR8 antagonists and mimicked by TLR7 but not TLR8 agonists. CVB3 infection of CD34+ cells increased the immunophenotype of MKs characterized as CD148+/CD48+ or CD41+/CD53+ cells. ConclusionThese data suggest a novel role of TLR7 in megakaryo/thrombopoiesis that may contribute to a better understanding of the molecular basis underlying thrombocytopenia and the immunologic role of MKs in viral infection processes.

Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms.

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient-derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders. This protocol was validated in: EMBO Rep (2022), DOI: 10.15252/embr.202152904 Graphical abstract Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation.

Low-Dose Rhein Sensitizes Platelet Production to Thrombopoietin Receptor Agonists (TPO-RA) in Immune Thrombocytopenia

Introduction: Primary immune thrombocytopenia (ITP) is an immune-mediated disorder characterized by isolated thrombocytopenia and risk of hemorrhage. Impaired megakaryocyte maturation and insufficient platelet formation, induced by platelet-specific autoantibodies or ITP plasma, play vital roles in its pathogenesis. Although thrombopoietin receptor agonists (TPO-RA) is recommended the first choice among second-line treatment of ITP, clinical data have indicated that nearly half of ITP patients failed or relapsed during eltrombopag maintenance, and increasing drug dose cannot reverse therapeutic response. Diacerein, hydrolyzed into its active metabolite rhein after oral administration, is a non-steroidal anti-inflammatory drug used in osteoarthritis. Our team first observed that 3 ITP patients with low platelet levels (30-50 ×109/L) during eltrombopag treatment, achieved significant platelet response (≥100×109/L) after taking diacerein concomitantly due to the complaint of arthralgia. These data suggested that diacerein may have sensitization effect on TPO-RA, and this novel combination is expected to boost thrombopoiesis in ITP. Thus, in vitro and in vivo experiments were performed to explore the role of TPO-RA plus rhein in megakaryocyte growth and platelet formation in ITP. Methods: Twenty-eight eltrombopag-inefficient or relapsed ITP patients and 15 healthy volunteers were included in this study. We sorted CD34+ cells from healthy umbilical cord blood mononuclear cells, then cultured them in SFEM medium containing thrombopoietin, interleukin-3, stem cell factor and 10% serum from either ITP patients or healthy donors. Different doses of eltrombopag combined with rhein (0-100 μM) were added on the fourth day, and cell suspension was collected on the 14th day. Flow cytometry, RNA sequencing, proteomic sequencing and cytokine assay were performed to evaluate the overlaying effect and underlying mechanisms of rhein on eltrombopag. In addition, active ITP murine models were established to evaluate the therapeutic effects of romiplostim plus rhein in vivo. Results: Compared with eltrombopag, eltrombopag combined with low-dose rhein significantly increased the proportion of CD41+, CD41+CD42+, CD41+CD61+ megakaryocytes in vitro. We further found that eltrombopag plus low-dose rhein significantly promoted polyploid maturation and apoptosis of CD41+ megakaryocytes, as well as platelet production in vitro (Fig 1). The ITP mice exhibited profound thrombocytopenia, and their platelet counts decreased significantly by the second week after irradiation. Our data showed that romiplostim plus low-dose rhein significantly alleviated thrombocytopenia than romiplostim monotherapy on day 21 and day 28 (Fig 2). Mice receiving different doses of rhein monotherapy were comparable in platelet counts with control group. Flow cytometry analysis suggested that the combination therapy significantly increased megakaryocyte percentage and polyploidization than romiplostim in ITP mice. Subsequently, RNA sequencing and proteomic analysis revealed that the combination of eltrombopag and low-dose rhein promoted megakaryocyte growth and platelet formation, decreased proinflammatory cytokines, and upregulated phosphorylated PI3K in vitro. Cytokine analysis indicated that eltrombopag plus low-dose rhein significantly increased supernatant level of TGF-β, and reduced IFN-α, IFN-γ, IL-12p70, IL-17A and IL-18 than eltrombopag. Furthermore, it was confirmed that the addition of rhein to TPO-RA significantly promoted megakaryocyte PI3K phosphorylation in vitro and in vivo. PI3K inhibition offset the enhanced effect of eltrombopag plus low-dose rhein over eltrombopag in megakaryocyte maturation and platelet formation. Conclusions: In sum, our data reveal a sensitizing role of rhein to TPO-RA in megakaryocyte polyploidization and platelet formation in ITP, and this novel combination therapy warrants further investigation as an optimized strategy for ITP patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigating ITP Plasma for Effect on Platelet Production Using the Autologous Peripheral Blood Based Megakaryopoiesis Assay

Investigating ITP plasma for effect on platelet production using the autologous peripheral blood based megakaryopoiesis assay Nikola Ivetic, Ishac Nazy, Rumi Claire, Angela Huynh John G. Kelton, Donald M. Arnold. Background: Studies focused on megakaryocyte number and function are needed to understand platelet underproduction as a potential mechanism of immune thrombocytopenia (ITP). Most experimental studies have relied on animal models, specialized cell lines, or exogenous and non-autologous sources of hematopoietic stem and progenitor cells (HSPCs) as a source of megakaryocytic cells and platelet production. We used HSPCs isolated from ITP patients themselves to examine the effects their plasma has megakaryocyte growth and platelet production. Study Design and Methods: Megakaryocytes were produced from peripheral blood CD34+ HSCs directly from patients with ITP and healthy controls. HSPC (CD45+ CD34+) were isolated, expanded for 4 days and then cultured with thrombopoietin (TPO) and stem cell factor (SCF) for 8 days. Megakaryocyte numbers were evaluated by flow cytometry using CD41a expression for megakaryopoiesis and CD42b expression for maturation. Platelet production was analyzed using CD41a expression and platelet forward and side scatter profile sizes. The addition of autologous ITP plasma and plasma depleted of IgG were also tested to determine the effects of antibodies on megakaryopoiesis and thrombopoiesis. Results: Peripheral blood derived megakaryocytes were cultured from HSPCs from ITP patients (N=10), and normal controls (N=9). Approximately 2,200±1,100 hematopoietic stem and progenitor cells were isolated per ml of peripheral blood and a 5-fold increase in stem cells was obtained after an expansion of the isolated ITP patient HSPC population. Following an additional 8 days of culture with TPO, 67±12% of the cultured cells were CD41a+ indicating they were undergoing megakaryopoiesis and 54±15% of the CD41a+ population expressed the CD42b marker for mature megakaryocytes. Of the 10 ITP cultures grown, four grew significantly more mature megakaryocytes in comparison to healthy controls. The addition of ITP plasma did not inhibit megakaryocyte growth or maturity, but platelet production was reduced with two samples demonstrating a 50% reduction in platelet levels in comparison to the . Relative to the number of mature megakaryocytes cultured there was a lower number of platelets detected in ITP cultures (3.34±1.9 platelets per mature megakaryocyte) in comparison to the healthy control group (8.1±1.4 platelets per mature megakaryocyte). Conclusions: The addition of patient plasma had no inhibiting effect on megakaryocyte counts in an autologous assay but a significant decrease in platelet number (thrombopoiesis) was detected. A subset of ITP HSPC generated more megakaryocytes when compared to healthy donors, indicating these cells are generating more megakaryocytes. This is to our knowledge the first autologous investigation of ITP plasma on patient HSPC undergoing megakaryopoiesis.

Association of Clinical Response of ITP Patients to Eltrombopag with the Regulation of Platelet Binding to Lymphocytes

Introduction Primary immune thrombocytopenia (ITP) is caused by the destruction of platelets (PLT) and megakaryocytes by autoantibodies, cellular cytotoxicity, or/and impairment in PLT production. Thrombopoietin-receptor agonists (TPO-RA) are a commonly used therapy in ITP that activate JAK/STAT pathways, stimulating megakaryocyte growth and PLT production. TPO-RAs, which include eltrombopag (EPAG), are effective in restoring PLT count in 70-80% of cases with chronic ITP. Recently, it has been observed that PLTs have an immunoregulatory role and their binding to lymphocytes and monocytes can modulate leukocyte inflammatory profile. Our aim was to determine the percentage of lymphocytes with bound PLTs before and after EPAG treatment, analyzing factors associated with this binding and with response. Methods For this purpose, peripheral whole blood was collected from 43 ITP patients relapsed/refractory to corticosteroids (at baseline and 2, 4, 12, and 18 w after the initiation of EPAG) and 15 healthy donors (HD). The percentage of T CD4+, T CD8+, and B lymphocytes with bound PLTs (CD41a+) were analyzed by flow cytometry and plasma levels of cytokines (IFN-γ, TNF-α, IL-10 and IL-17) by ELISA. Results In comparison with HD, the absolute number of PLTs was lower and the percentage of free activated PLTs was higher in ITP patients at baseline. Additionally, the percentage of T and B lymphocytes with bound PLTs in ITP patients was lower than in HD. After 2w of EPAG treatment, the percentages of T and B lymphocytes with bound PLTs were normalized and IFN-g levels were reduced. However, no significant changes were observed in the percentage of free-activated PLTs and in the percentage of T and B lymphocytes with bound activated PLTs. The percentages of T and B lymphocytes with bound PLTs were higher in ITP patients that reached complete response (>100 x 109 PLT/L without bleeding) compared with partial response (30-100 x 109 PLT/L) and non-response (<30x 109 PLT/L) patients in each time point of the study after EPAG treatment (Figure 1). Conclusions Our results suggest that the increase of PLTs after EPAG treatment is not only mediated through the platelet production stimulation, but also through the increase of PLT binding to lymphocytes which may influence the inflammatory profile of ITP patients. This newly mechanism of action could explain the persistent response of ITP patients after EPAG treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IL-13/IL-4 signaling contributes to fibrotic progression of the myeloproliferative neoplasms

AbstractMyelofibrosis (MF) is a disease associated with high unmet medical needs because allogeneic stem cell transplantation is not an option for most patients, and JAK inhibitors are generally effective for only 2 to 3 years and do not delay disease progression. MF is characterized by dysplastic megakaryocytic hyperplasia and progression to fulminant disease, which is associated with progressively increasing marrow fibrosis. Despite evidence that the inflammatory milieu in MF contributes to disease progression, the specific factors that promote megakaryocyte growth are poorly understood. Here, we analyzed changes in the cytokine profiles of MF mouse models before and after the development of fibrosis, coupled with the analysis of bone marrow populations using single-cell RNA sequencing. We found high interleukin 13 (IL-13) levels in the bone marrow of MF mice. IL-13 promoted the growth of mutant megakaryocytes and induced surface expression of transforming growth factor β and collagen biosynthesis. Similarly, analysis of samples from patients with MF revealed elevated levels of IL-13 in the plasma and increased IL-13 receptor expression in marrow megakaryocytes. In vivo, IL-13 overexpression promoted disease progression, whereas reducing IL-13/IL-4 signaling reduced several features of the disease, including fibrosis. Finally, we observed an increase in the number of marrow T cells and mast cells, which are known sources of IL-13. Together, our data demonstrate that IL-13 is involved in disease progression in MF and that inhibition of the IL-13/IL-4 signaling pathway might serve as a novel therapeutic target to treat MF.

Megakaryopoiesis impairment through acute innate immune signaling activation by azacitidine.

Thrombocytopenia, prevalent in the majority of patients with myeloid malignancies, such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), is an independent adverse prognostic factor. Azacitidine (AZA), a mainstay therapeutic agent for stem cell transplant-ineligible patients with MDS/AML, often transiently induces or further aggravates disease-associated thrombocytopenia by an unknown mechanism. Here, we uncover the critical role of an acute type-I interferon (IFN-I) signaling activation in suppressing megakaryopoiesis in AZA-mediated thrombocytopenia. We demonstrate that megakaryocytic lineage-primed progenitors present IFN-I receptors and, upon AZA exposure, engage STAT1/SOCS1-dependent downstream signaling prematurely attenuating thrombopoietin receptor (TPO-R) signaling and constraining megakaryocytic progenitor cell growth and differentiation following TPO-R stimulation. Our findings directly implicate RNA demethylation and IFN-I signal activation as a root cause for AZA-mediated thrombocytopenia and suggest mitigation of TPO-R inhibitory innate immune signaling as a suitable therapeutic strategy to support platelet production, particularly during the early phases of AZA therapy.

Apoptosis of Megakaryocytic Leukemia Cell Line Meg-01 Induced by TSP-1 Via CD36/Caspase-3

To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture. The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 μg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 μg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 μg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 μg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 μg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while β-TG could not. CD36 antibody FA6-152 (25 μg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK. TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.

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.

Treatment of chemotherapy-induced thrombocytopenia in patients with non-hematologic malignancies.

Chemotherapy-induced thrombocytopenia (CIT) is a common complication of the treatment of non-hematologic malignancies. Many patient-related variables (e.g., age, tumor type, number of prior chemotherapy cycles, amount of bone marrow tumor involvement) determine the extent of CIT. CIT is related to the type and dose of chemotherapy, with regimens containing gemcitabine, platinum, or temozolomide producing it most commonly. Bleeding and the need for platelet transfusions in CIT are rather uncommon except in patients with platelet counts below 25x109/L in whom bleeding rates increase significantly and platelet transfusions are the only treatment. Nonetheless, platelet counts below 70x109/L present a challenge. In patients with such counts, it is important to exclude other causes of thrombocytopenia (medications, infection, thrombotic microangiopathy, post-transfusion purpura, coagulopathy and immune thrombocytopenia). If these are not present, the common approach is to reduce chemotherapy dose intensity or switch to other agents. Unfortunately decreasing relative dose intensity is associated with reduced tumor response and remission rates. Thrombopoietic growth factors (recombinant human thrombopoietin, pegylated human megakaryocyte growth and development factor, romiplostim, eltrombopag, avatrombopag and hetrombopag) improve pretreatment and nadir platelet counts, reduce the need for platelet transfusions, and enable chemotherapy dose intensity to be maintained. National Comprehensive Cancer Network guidelines permit their use but their widespread adoption awaits adequate phase III randomized, placebo-controlled studies demonstrating maintenance of relative dose intensity, reduction of platelet transfusions and bleeding, and possibly improved survival. Their potential appropriate use also depends on consensus by the oncology community as to what constitutes an appropriate pretreatment platelet count as well as identification of patient-related and treatment variables that might predict bleeding.

Megakaryocyte–stromal cell interactions: Effect on megakaryocyte proliferation, proplatelet production, and survival

Bone marrow stromal cells provide a proper environment for the development of hematologic lineages. The incorporation of different stromal cells into in vitro culture systems would be an attractive model to study megakaryopoiesis and thrombopoiesis. Our objective was to evaluate the participation of different types of stromal cells in in vitro megakaryopoiesis, thrombopoiesis, and megakaryocyte (MK) survival. CD34-positive progenitors from umbilical cord blood were differentiated into MK precursors and then cocultured with umbilical vein endothelial cells (HUVECs), bone marrow mesenchymal stem cells (MSCs), skin fibroblasts (SFs) (all human), or the mouse fibroblast cell line L929. The number of MKs (CD61-positive cells) was increased in the presence of HUVECs and SFs, whereas L929 cells decreased total and mature MK counts. With respect to thrombopoiesis, HUVECs increased proplatelet (PP)-producing MKs, while MSCs, L929 cells, and SFs had the opposite effect (immunofluorescence staining and microscopic analysis). MK survival was enhanced in MSC and SF co-cultures, as assessed by evaluation of pyknotic nuclei. However, HUVECs and L929 did not prevent apoptosis of MKs. Reciprocally, the cloning efficiency of MSCs was decreased in the presence of MKs, while the ability of stromal cells (either MSCs or SFs) to produce the extracellular matrix proteins type III collagen, fibronectin, dermatan sulfate, heparan sulfate, and prolyl 4-hydroxylase subunit β was preserved. These data indicate that each stromal cell type performs distinctive functions that differentially modulate MK growth and platelet production and, at the same time, that MKs also modify stromal cell behavior. Overall, our results highlight the relevance of considering the influence of stromal cells in MK research as well as the close interplay of different cell types within the bone marrow milieu.

THROMBOTIC MICROANGIOPATHIC HEMOLYTIC ANEMIA IN SYSTEMIC LUPUS ERYTHEMATOSUS IN A 25 YEARS OLD FEMALE IN CHENNAI, INDIA

Systemic lupus erythematosus (SLE) is an autoimmune condition, affecting multiple organs and tissues by autoantibodies and immune complexes. Hematologic manifestations of SLE include anemia, thrombocytopenia and leukopenia. The most common type of anemia seen in SLE is anemia of chronic disease. Thrombotic microangiopathic hemolytic anemia (TMHA) in SLE is very rare. Incidence of TMHA in children with SLE is common compared to adult cases of SLE. Manifestations of TMHA include presence of schistocytes in the peripheral smear, elevated bilirubin levels, elevated LDH and negative coombs test. This case report focuses on 25-year-old female diagnosed concomitantly with SLE and TMHA, and treated with steroids, blood transfusions, megakaryocyte growth factors and antibiotics.

The Effects of TMP Treatment and High Fat Diet on Bone Fracture Healing

Delayed and impaired bone fracture healing are associated with diabetic populations. This is a challenging problem for orthopaedic surgeons especially in the US where the percentage of type 2 diabetic patients continues to climb at an alarming rate. Limited treatment options exist for orthopaedic surgeons to improve fracture healing, and the most commonly used therapies involve placement of proteins (bone morphogenetic protein), graft tissue, or demineralized bone matrix at the fracture site. We have previously demonstrated that local administration of the main megakaryocyte growth factor, thrombopoietin, enhances bone healing. Here we demonstrate the utility of systemically administering thrombopoietin mimetic peptides (TMPs) to improve impaired fracture healing in a mouse model of type 2 diabetes. Briefly, 120 male mice on a C57BL/6 background were placed on a low fat diet (LFD) or high fat diet (HFD) for 12 weeks prior to undergoing a surgically created femoral fracture. Mice were treated with 33 nmol/kg of TMP or saline immediately after surgery and daily for the following week. Mice were euthanized at 1, 2, and 4 weeks post-surgery (n=10/group). Here, we confirmed that HFD resulted in impaired fracture healing. We also showed accelerated bone union and increased callus formation in TMP treated mice compared to saline groups, irrespective of diet (p&lt;0.05). Among TMP groups that were fed either a HFD or LFD, the HFD TMP group showed greater improvements in bone healing compared to the HFD saline control mice. Further study on TMP should include alternative routes of administration and providing treatment when a surgical repair appears to be deteriorating. Although there is more to be understood about the clinical importance and mechanism by which systemic TMP treatment enhances fracture healing, these data appear promising.

Effect of Thrombospondin-1 on Apoptosis of Human Megakaryocytic Leukemia Cells

Background: Thrombospondin 1 (TSP-1) is an extracellular matrix protein that interacts with a wide array of ligands including cell receptors, growth factors, cytokines, and proteases to regulate various physiological and pathological processes. TSP-1 induces apoptosis of endothelial and cancer cells via its receptor CD36. This study was to investigate the effect of TSP-1 on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism.Methods: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations, to investigate the effect of TSP-1 on the growth of megakaryocytes. The early apoptosis and the activity of the apoptotic protease caspase-3 were detected by flow cytometry. Bone marrow cells of mice were cultured for CFU-MK and stained with acetylcholine to detect the differentiation of megakaryocytes.Results: CD36 antigen was detected on the surface of Meg-01 cells by flow cytometry and immunocytochemistry. TSP-1 (5 μg/mL) inhibited the proliferation of Meg-01 cells, but not M-07e cells (CD36 -). After the addition of CD36 antibody FA6-152 (5, 10 and 25 μg/mL), the inhibitory effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5 and 7.5 μg/mL) exerted a pro-apoptotic effect by increasing the expression of Annexin V (P<0.01) and caspase-3 activation (P<0.01). Addition of FA6-152 (25 μg/mL) can significantly reduce the apoptosis induced by TSP-1 in Meg-01 cells. In addition, TSP-1 (5, 10 and 25 μg/mL) repress the formation of CFU-MK in mouse bone marrow cells, while β-TG did not. This repression was relieved efficiently by FA6-152 (25 μg/mL).Conclusion: TSP-1 could inhibit the proliferation of Megakaryocyte cell line Meg-01 cells, and induce it's apoptosis. TSP-1 may induce apoptosis of Meg-01 cells via CD36 or caspase-3, which provides a potential new drug choice for clinical treatment of megakaryocytic leukemia. DisclosuresNo relevant conflicts of interest to declare.

TPO versus TPO: the good, the bad, and the to be determined.

TPO versus TPO: the good, the bad, and the to be determined.

The megakaryocytic transcription factor ARID3A suppresses leukemia pathogenesis

AbstractGiven the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21–encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.

Induced Pluripotent Stem Cells Enable Disease Modeling and Drug Screening in Calreticulin del52 and ins5 Myeloproliferative Neoplasms

Mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and primary myelofibrosis patients. To address the contribution of the human CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) in an endogenous context, we modeled the CALRdel52 and CALRins5 mutants by induced pluripotent stem cell (iPSC) technology using CD34+ progenitors from 4 patients. We describe here the generation of several clones of iPSC carrying heterozygous CALRdel52 or CALRins5 mutations. We showed that CALRdel52 induces a stronger increase in progenitors than CALRins5 and that both CALRdel52 and CALRins5 mutants favor an expansion of the megakaryocytic lineage. Moreover, we found that both CALRdel52 and CALRins5 mutants rendered colony forming unit–megakaryocyte (CFU-MK) independent from thrombopoietin (TPO), and promoted a mild constitutive activation level of signal transducer and activator of transcription 3 in megakaryocytes. Unexpectedly, a mild increase in the sensitivity of colony forming unit-granulocyte (CFU-G) to granulocyte-colony stimulating factor was also observed in iPSC CALRdel52 and CALRins5 compared with control iPSC. Moreover, CALRdel52-induced megakaryocytic spontaneous growth is more dependent on Janus kinase 2/phosphoinositide 3-kinase/extracellular signal-regulated kinase than TPO-mediated growth and opens a therapeutic window for treatments in CALR-mutated MPN. The iPSC models described here represent an interesting platform for testing newly developed inhibitors. Altogether, this study shows that CALR-mutated iPSC recapitulate MPN phenotypes in vitro and may be used for drug screening.

Developing Live Imaging Strategies to Analyze the Influence of Cell Cycle Rate on MEP Fate Decisions at the Single Cell Level

Elucidating the mechanisms underlying hematopoietic cell fate decision is critical to our understanding of physiological and pathological hematopoiesis. Robust response to physiological stress requires rapid commitment and expansion of mature blood cells derived from less committed progenitors. The processes dictating the directed differentiation of these progenitors are poorly understood.Preliminary data from our lab suggest that cell cycle rate influences commitment of the Megakaryocyte/Erythroid Progenitor (MEP) to the megakaryocytic and erythroid (Er) lineages. To test whether slower cycling MEPs tend to be more committed to the megakaryocytic lineage, we have developed a time-lapse microscopy imaging approach to measure the number and duration of cell cycles of single cells and their progeny as they make their cell fate decision determined by their fluorescent marker expression in real-time.CD34-selected mobilized peripheral blood cells were stained with desired antibodies and sorted using our recently published scheme: Lin-CD34+CD38midCD45RA-FLT3-MPL+CD36-CD41- (Sanada and Xavier-Ferrucio, et al. Blood 2016) that is highly enriched for bipotent MEPs than populations previously reported. MEPs were plated at low density (25 cells/15μL) in MegaCult collagen-based medium containing SCF, IL3, IL6, TPO, and EPO in the center of a MatTek plate under a coverslip to minimize focal range. After the MegaCult polymerized, 1.5mL of additional MegaCult and cytokines were added to the dish to support growth and expansion of megakaryocytes and erythroid cells for 12 days. Cells were imaged every 2 hours in DIC for the first three days, and every 20 minutes for 8 additional days. On the 10th day, fluorescently conjugated anti-CD41 (to identify Mks) and anti-CD235a (to identify Er cells) antibodies were diluted in IMDM and added to the imaged dish. The antibodies permeated the MegaCult and labeled the committed cells. Imaging continued every 20 minutes in DIC, FITC, and Cy5 channels for an additional day. Using the Baxter Algorithm to analyze time-lapse images, we traced the progeny back to the parent cell, and calculated the number of cell cycles, as well as the duration of each cycle leading to lineage commitment. We have validated this method by measuring cell cycle rates of bi-potent MEPs, Mk-restricted progenitors, and Er-restricted progenitors, and found Er-restricted progenitors to have a significantly shortened cell cycle rate compared to Mk-restricted progenitors.To further investigate the role of cell cycle rate in MEP commitment, we utilized our live imaging strategy to collect and analyze cell cycle rate data for transduced MEPs expressing shRNA against Myb. Myb is a known regulator of the G2/M transition by binding the promoter and altering expression of Cyclin B1 in human blood cells (Nakata et al., MCB, 2007). Here we show that MEPs knocked down for Myb have no difference in survival compared to scramble controls. Interestingly, the rate of cell cycle divisions is significantly longer in MEP progeny lacking Myb, and this correlates with an increase in the ratio of CFU-Mk to BFU-E and CFU-Mk/E. This data suggests that by lengthening the cell cycle rate, fate decision of the bipotent MEP can be swayed towards the Mk lineage, which has a longer cell cycle rate compared to the Er lineage. In the future, live imaging analysis of single cells can be utilized to answer a plethora of questions regarding fate decisions and lineage commitment of hematopoietic stem and progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Gaussian Mixture Models and Machine Learning Predict Megakaryocytic Growth and Differentiation Potential Ex Vivo

The ability to analyze single cells via flow cytometry has resulted in a wide range of biological and medical applications, such as monitoring populations of developing immune and hematopoietic cells over time. Currently, there is no established framework to compare and interpret time-series flow cytometry data for cellular engineering applications. Manual analysis of temporal trends is time-consuming and subjective for large-scale datasets. We resolved this bottleneck by developing TEmporal Gaussian Mixture models (TEGM), an unbiased computational strategy to quantify and predict temporal trends of developing cell subpopulations indicative of cellular phenotype. We validated its utility by accurately predicting peak CD41+/CD42+ % of megakaryocyte (MK) cultures derived from human CD34+-selected umbilical cord blood units (CB).TEGM applies Gaussian mixture modeling for feature extraction and gradient boosted trees for prediction. Our modeling strategy allows for data to be gated and clustered on multi-dimensional planes, enabling rapid processing of flow cytometry data. TEGM enables the extraction of subtle features, such as the dispersion and rate of change of surface marker expression for each subpopulation over time. These critical, yet hard-to-discern, features are fed into machine-learning algorithms that predict underlying cellular classes and phenotypes. Our framework can be flexibly applied to conventional flow cytometry sampling schemes, and allows for faster and more consistent processing of time-series flow cytometry data.As proof-of-concept, we applied our method to the analysis of ex vivo MK differentiation and maturation of hematopoietic cells from donors with varying potential to generate CD41+/CD42+ cells. Our computational approach consists of three major steps: preprocessing, feature extraction, and prediction (Figure 1). We illustrate the major steps of the computational approach by predicting peak CD41+/CD42+ % MK maturation of CD34+-selected CB cells from 16 independent donors. Cells were cultured over a 19-day multi-phase differentiation culture, consisting of a pre-expansion phase and a differentiation phase. The novel dataset comprised 720 measurements from 80 perturbations of 16 individual donors, with 9 time-point measurements sampled every 2-5 days for each donor. In the preprocessing step, we filtered out non-uniform events, margin events, and doublets. We then constructed an automated gating strategy to extract surface marker expression of various clusters of DAPIlow/CD41+ MK cells. Notably, we demonstrated that estimation of the CD34+ and CD42+ % was within 1% of manual gating estimates, thus illustrating the consistency and accuracy of the technique. We then performed feature extraction for each flow cytometry time-course dataset on several descriptors, such as growth rate, viability, production, percentage positivity of each surface marker, covariance of mean fluorescence intensity, rate of change, and bifurcation of each subpopulation. A gradient boosted tree model was trained using an explanatory matrix describing early characteristics (Day 0-Day 9) and tested to predict peak CD41+/CD42+ marker expression, which typically occurs on Day 14 to Day 17.Overall, we identified several influential early culture factors that are predictive of peak CD42+ % expression. We showed that CD41+ % on Day 5 and Day 7 is highly predictive of peak CD42+ % expression. Cell viability and CD34+ % were comparatively less predictive of peak CD42+ %. Based on our analysis, we were able to identify the best performing cultures with high sensitivity and specificity (AUROC = 0.92, where 1 denotes perfect accuracy). Predicted and actual CD41+/CD42+ responses were highly correlated (p=7.4e09, R = 0.87) using 3 independently selected partitions of test/training sets of our data. We demonstrated that the resulting model captured apparent cell development processes represented by cell surface markers including CD34, CD41, and CD42, and was able to predict MK differentiation and maturation potential of a given CB unit in culture. Identifying CB units with high and low MK potential early in the 19-day culture process can save expensive resources and time, and provides the potential to intervene during the culture process.Figure 1. Manual gating strategy (left) vs. TEGM gating strategy (right) for time-series flow cytometry data. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.