Platelet enhancement by Carica papaya L. leaf fractions in cyclophosphamide induced thrombocytopenic rats is due to elevated expression of CD110 receptor on megakaryocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Carica papaya leaf juice/decoction has been in use in folk medicine in Srilanka, Malaysia and in few parts of India for enhancing the platelet counts in dengue. In Siddha medicine, a traditional form of medicine in India, papaya leaf juice has been used for increasing the platelet counts. Papaya leaf has been reported to enhance blood volume in ancient Ayurveda books in India. Carica papaya leaf is well known for its platelet enhancement activity. Although many preclinical and clinical studies have demonstrated the ability of papaya leaf juice for platelet enhancement, but the underlying mechanisms are still unclear. AIM OF THE STUDY: The study is aimed at identifying the key ingredients of papaya leaf extract and elucidate the mechanism (s) of action of the identified potent component in mitigating thrombocytopenia (Thp). MATERIALS AND METHODS: C. papaya leaf juice was subjected for sequential fractionation to identify the anti-thrombocytopenic phytochemicals. In vivo, stable thrombocytopenia was induced by subcutaneous injection of 70 mg/kg cyclophosphamide (Cyp). After induction, rats were treated with 200 and 400 mg/kg body weight papaya leaf juice and with identified fractions for 14 days. Serum thrombopoietin level was estimated using ELISA. CD110/cMpl, a receptor for thrombopoietin on platelets was measured by western blotting. RESULTS: Administration of cyclophosphamide for 6 days induced thrombocytopenia (210.4 ± 14.2 × 103 cells/µL) in rats. Treating thrombocytopenic rats with papaya leaf juice and butanol fraction for 14 days significantly increased the platelet count to 1073.50 ± 29.6 and 1189.80 ± 36.5 × 103 cells/µL, respectively. C.papaya extracts normalized the elevated bleeding and clotting time and decreased oxidative markers by increasing endogenous antioxidants. A marginal increase in the serum thrombopoietin (TPO) level was observed in Cyp treated group compared to normal and treatment groups. Low expression of CD110/cMpl receptor found in Cyp treated group was enhanced by C. papaya extracts (CPJ) and CPJ-BT. Furthermore, examination of the morphology of bone marrow megakaryocytes, histopathology of liver and kidneys revealed the ability of CPJ and fractions in mitigating Cyp-induced thrombocytopenia in rats. CONCLUSION: C. papaya leaf juice enhances the platelet count in chemotherapy-induced thrombocytopenia by increasing the expression of CD110 receptor on the megakaryocytes. Hence, activating CD110 receptor might be a viable strategy to increase the platelet production in individuals suffering from thrombocytopenia.

A ß1-tubulin-based megakaryocyte maturation reporter system identifies novel drugs that promote platelet production.

During maturation, megakaryocytes (MKs) express ß1-tubulin (TUBB1) and rearrange their microtubule components to enlarge, form proplatelets, and eventually release platelets. The development of a platform to identify in vitro conditions that would efficiently promote MK development could potentially enable large-scale platelet production. Here, we show that an immortalized MK cell line (imMKCL) genetically modified to express the ß1-tubulin-Venus reporter provides a practical system to efficiently monitor the in vitro production of platelet-like particles (PLPs). The Venus transgene was inserted downstream of the TUBB1 locus in imMKCLs using CRISPR/Cas9, and the expression was visualized by Venus fluorescence intensity. This imMKCL reporter line was then used for high-throughput drug screening. We identified several compounds that significantly improved the efficiency of PLP production in vitro under feeder-free conditions and showed a significant tendency to recover platelets in vivo in a mouse thrombocytopenia model induced by anti-GPIbα antibody administration. Interestingly, most of these compounds, including a WNT signaling pathway inhibitor, Wnt-C59, antagonized the aryl hydrocarbon receptor (AhR) to increase PLP production, confirming the crucial role of AhR inhibition in MK maturation. Consistently, small interfering RNA treatment against AhR increased the Venus intensity and PLP production. TCS 359, an FLT3 inhibitor, significantly increased PLP production independently of FLT3 or AhR. This study highlights the usefulness of the ß1-tubulin reporter MK line as a useful tool to study the mechanisms underlying thrombopoiesis and to identify novel inducers of ex vivo platelet production.

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

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

Investigation of Iron Metabolism for Regulating Megakaryopoiesis and Platelet Count According to the Mechanisms of Anemia.

BACKGROUND: Iron deficiency anemia (IDA) is characterized by depletion of total body iron stores or a poor supply of plasma iron. By contrast, chronic inflammation makes iron unavailable for hematopoiesis through a cytokinemediated cascade and leads to a condition known as anemia of chronic disease (AOC). However, the laboratory data regarding the regulatory role of iron metabolism on platelet count has not been fully discussed yet. In this study, we investigated the relationship between iron status and platelet production according to different anemic mechanisms representing different iron metabolisms. METHODS: The study included a total of 759 specimens. Blood samples were obtained through venipuncture. The complete blood count was measured using an Advia 2120 (Siemens Healthcare Diagnostics Inc., USA). Biochemical indices including iron level were estimated using a Toshiba chemical analyzer (Toshiba, Japan). RESULTS: In the AOC group, we found a significant relationship between platelet count and serum iron level (p < 0.27), whereas there was no correlation in the IDA group. Moreover, when the AOC patient group was subdivided by serum iron level, a remarkable difference was observed as follows. The platelet count was significantly correlated with serum iron level only in the AOC group with decreased serum iron levels (serum iron < 50 µg/dL) (p < 0.0001), while there was no correlation in the AOC group with normal serum iron levels (serum iron 50 - 100 µg/dL). CONCLUSIONS: Iron deficiency in AOC involves upregulated hepcidin production induced by elevated inflammatory cytokines. This can cause increased iron sequestration in macrophages and decreased iron absorption for bone marrow. The condition of decreased megakaryocytic iron supply makes megakaryocytes with higher ploidy which can release more platelets than lower ploidy. Moreover, reactive thrombocytosis in inflammatory states occurs by cytokine cascades involving interleukin 6 and thrombopoietin in AOC. These two features may enhance thrombocytosis in patients of AOC with decreased iron level. In the future, further study should be performed to elucidate regulating mechanism of iron metabolism for megakaryopoiesis in AOC patients, and guide proper supplemental therapy of iron to decrease thrombotic risk due to reactive thrombocytosis in various kinds of anemia.

Justicia adhatoda induces megakaryocyte differentiation through mitochondrial ROS generation.

BACKGROUND: Hepatoprotective activity along with improved survival percentage and hematological parameters prior to whole body irradiation were reported with Justicia adhatoda extracts. PURPOSE: To evaluate the thrombopoietic potential of Justicia adhatoda L. leaf extract in megakaryocyte differentiation METHODS: Ethanol extracts were prepared using soxhlet extraction method, and IC50 value was determined. The effect of ethanol extracts obtained from Justicia adhatoda on megakaryocyte maturation and development in megakaryocytic Dami cell lines was tested. Expression of megakaryocyte specific markers, CD61 and CD41, were assessed using flow cytometry and fluorescence microscopy. In addition, cell cycle analysis and mitochondrial membrane potential were analyzed by flow cytometry. Gene expression analysis was performed using qRT-PCR. RESULTS: At a concentration of 40 µg/ml, the leaf extracts of Justicia adhatoda for 72 h induced the megakaryocytic features in megakaryocytic Dami cell lines. The megakaryocyte specific markers, CD41 and CD61, were up-regulated (2.2 and 12.4 fold, respectively), and more number of cells entered into synthetic (S) and G2/M phase as compared with untreated cell (23.1% vs 16.6% and 70.2% vs 42.3%, respectively) showing maturation. RUNX1 (a transcription factor essential for embryonic hematopoiesis and adult megkaryocyte maturation) and c-Mpl (the receptor for TPO) were upregulated, and the suppressor of cytokine signaling (SOCS) 1 and SOCS3 were down-regulated upon treatment with Justicia adhatoda. Justicia adhatoda enhanced mitochondrial ROS generation by 28-fold, increased the permeability of mitochondrial membrane and showed an inverse correlation in superoxide dismutase levels. CONCLUSION: Justicia adhatoda could enhance mitochondrial ROS generation and increase the permeability of mitochondrial membrane, thereby inducing megakaryocytic maturation. Our findings suggest thrombopoietic potential of Justicia adhatoda leaf extract on megakaryocyte differentiation.

R-lipoic acid overdosing affects platelet life span via ROS mediated autophagy.

R-lipoic acid (ALA), a powerful antioxidant valuable for the treatment of diabetes and its complications, has been reported to exhibit an antiplatelet activity in vitro. The aim of this study was to investigate the effect and mechanism of ALA on platelets in vivo. Sprague-Dawley (SD) male rats were intravenously administered with low-dose ALA (20 mg/kg/d), high-dose ALA (80 mg/kg/d) and saline, respectively. Platelets count and bone marrow smear were evaluated and the expressions of markers related to apoptosis and autophagy were measured. Platelet clearance analysis was conducted out on mice. The results showed that high-dose ALA administration could significantly decrease platelet count by 43% compared with control group, whereas, megakaryocytes showed no difference in the number. Moreover, high-dose ALA administration led to significant reduction in half-life of circulating platelets, indicative of enhanced rate of platelet clearance. Interesting, high-dose ALA administration could increase the level of reactive oxygen species (ROS) in platelets and induce autophagy without affecting apoptosis. Our finding also showed that high ALA-induced autophagy in platelets was mediated by class III PtdIns3K activity, which could be reversed by 3-methyladenine (3-MA). Moreover, AKT and MAPK/ERK pathways were also observed to be involved in the regulation of autophagy in platelets. Thus, high-dose ALA could induce autophagy in platelets through modulating the activity of class III PtdIns3K, which was associated with decreased count of circulating platelets and shortened lifespan of platelets.

A novel mode of stimulating platelet formation activity in megakaryocytes with peanut skin extract.

We report in this study novel biochemical activities of peanut skin extract (PEXT) on thrombocytopoiesis. Peanut skin, derived from Arachis hypogaea L., is a traditional Chinese medicine that is used to treat chronic hemorrhage. We have shown that oral administration of PEXT increases the peripheral platelet levels in mice. Recently, we reported a liquid culture system that is useful for investigating megakaryocytopoiesis and thrombocytopoiesis from human CD34+ cells. In this liquid culture system, PEXT was shown to enhance the formation of CD41+/DAPI- cells (platelets), but had no effect on the formation of CD41+/DAPI+ cells (megakaryocytes) or on the DNA content. Furthermore, PEXT selectively stimulated proplatelet formation from cultured mature megakaryocytes and phorbol 12-myristate 13 acetate (PMA)-induced formation of platelet-like particles from Meg01 cells. Despite having no influence on the formation of megakaryocyte colony forming units (CFUs), PEXT increased the size of megakaryocytes during their development from CD34+ cells. PEXT showed no effect on the GATA-1 and NF-E2 mRNA levels, which are known to play an important role in thrombocytopoiesis and, based on the results of a pMARE-Luc (pGL3-MARE-luciferase) assay, had no influence on NF-E2 activation in Meg01 cells. These results suggest that PEXT accelerates proplatelet formation from megakaryocytes but does not influence the development of hematopoietic stem cells into megakaryocytes.

Development of a new knock-in mouse model and evaluation of pharmacological activities of lusutrombopag, a novel, nonpeptidyl small-molecule agonist of the human thrombopoietin receptor c-Mpl.

Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production.

Small Molecule Supplements Improve Cultured Megakaryocyte Polyploidization by Modulating Multiple Cell Cycle Regulators.

Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications.

The contribution of mouse models to the understanding of constitutional thrombocytopenia.

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.

Noninvasive low-level laser therapy for thrombocytopenia.

Thrombocytopenia is a common hematologic disorder that is managed primarily by platelet transfusions. We report here that noninvasive whole-body illumination with a special near-infrared laser cures acute thrombocytopenia triggered by γ-irradiation within 2 weeks in mice, as opposed to a 5-week recovery time required in controls. The low-level laser (LLL) also greatly accelerated platelet regeneration in the presence of anti-CD41 antibody that binds and depletes platelets, and prevented a severe drop in platelet count caused by a common chemotherapeutic drug. Mechanistically, LLL stimulated mitochondrial biogenesis specifically in megakaryocytes owing to polyploidy of the cells. LLL also protected megakaryocytes from mitochondrial injury and apoptosis under stress. The multifaceted effects of LLL on mitochondria bolstered megakaryocyte maturation; facilitated elongation, branching, and formation of proplatelets; and doubled the number of platelets generated from individual megakaryocytes in mice. LLL-mediated platelet biogenesis depended on megakaryopoiesis and was inversely correlated with platelet counts, which kept platelet biogenesis in check and effectively averted thrombosis even after repeated uses, in sharp contrast to all current agents that stimulate the differentiation of megakaryocyte progenitors from hematopoietic stem cells independently of platelet counts. This safe, drug-free, donor-independent modality represents a paradigm shift in the prophylaxis and treatment of thrombocytopenia.

Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg(2+) homeostasis and cytoskeletal architecture.

Mg(2+) plays a vital role in platelet function, but despite implications for life-threatening conditions such as stroke or myocardial infarction, the mechanisms controlling [Mg(2+)]i in megakaryocytes (MKs) and platelets are largely unknown. Transient receptor potential melastatin-like 7 channel (TRPM7) is a ubiquitous, constitutively active cation channel with a cytosolic α-kinase domain that is critical for embryonic development and cell survival. Here we report that impaired channel function of TRPM7 in MKs causes macrothrombocytopenia in mice (Trpm7(fl/fl-Pf4Cre)) and likely in several members of a human pedigree that, in addition, suffer from atrial fibrillation. The defect in platelet biogenesis is mainly caused by cytoskeletal alterations resulting in impaired proplatelet formation by Trpm7(fl/fl-Pf4Cre) MKs, which is rescued by Mg(2+) supplementation or chemical inhibition of non-muscle myosin IIA heavy chain activity. Collectively, our findings reveal that TRPM7 dysfunction may cause macrothrombocytopenia in humans and mice.

Effects of panaxadiol saponins component as a new Chinese patent medicine on proliferation, differentiation and corresponding gene expression profile of megakaryocytes.

OBJECTIVE: To investigate the effects of panaxadiol saponins component (PDS-C) isolated from total saponins of panax ginseng on proliferation, differentiation and corresponding gene expression profile of megakaryocytes. METHODS: Bone marrow culture of colony forming assay of megakaryocytic progenitor cells (CFU-MK) was observed for the promoting proliferation mediated by PDS-C, and differentiation of megakaryocytic blasts caused by PDS-C was analyzed with flow cytometry in CHRF-288 and Meg-01 cells, as well as proliferation, differentiation-related genes expression profile and protein expression levels were detected by human gene expression microarray and western blot. RESULTS: In response to PDS-C 10, 20 and 50 mg/L, CFU-MK from 10 human bone marrow samples was increased by 28.9%±2.7%, 41.0%±3.2% and 40.5%±2.6% over untreated control, respectively (P <0.01, each). Flow cytometry analysis showed that PDS-C treated CHRF-288 cells and Meg-01 cells significantly increased in CD42b, CD41, TSP and CD36 positive ratio, respectively. PDS-C induced 29 genes up-regulated more than two-fold commonly in both cells detected by human expression microarray representing 4000 known genes. The protein expression levels of ZNF91, c-Fos, BTF3a, GATA-1, RGS2, NDRG2 and RUNX1 were increased with western blot in correspond to microarray results. CONCLUSION: PDS-C as an effective component for hematopoiesis, play the role to enhance proliferation and differentiation of megakaryocytes, also up-regulated expression of proliferation, differentiation-related genes and proteins in vitro.

Preclinical models of nicotinamide phosphoribosyltransferase inhibitor-mediated hematotoxicity and mitigation by co-treatment with nicotinic acid.

Nicotinamide adenine dinucleotide (NAD) is an essential co-factor in glycolysis and is a key molecule involved in maintaining cellular energy metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of an important salvage pathway in which nicotinamide is recycled into NAD. NAMPT is up-regulated in many types of cancer and NAMPT inhibitors (NAMPTi) have potential therapeutic benefit in cancer by impairing tumor metabolism. Clinical trials with NAMPTi APO-866 and GMX-1778, however, failed to reach projected efficacious exposures due to dose-limiting thrombocytopenia. We evaluated preclinical models for thrombocytopenia that could be used in candidate drug selection and risk mitigation strategies for NAMPTi-related toxicity. Rats treated with a suite of structurally diverse and potent NAMPTi at maximum tolerated doses had decreased reticulocyte and lymphocyte counts, but no thrombocytopenia. We therefore evaluated and qualified a human colony forming unit-megakaryocyte (CFU-MK) as in vitro predictive model of NAMPTi-induced MK toxicity and thrombocytopenia. We further demonstrate that the MK toxicity is on-target based on the evidence that nicotinic acid (NA), which is converted to NAD via a NAMPT-independent pathway, can mitigate NAMPTi toxicity to human CFU-MK in vitro and was also protective for the hematotoxicity in rats in vivo. Finally, assessment of CFU-MK and human platelet bioenergetics and function show that NAMPTi was toxic to MK and not platelets, which is consistent with the clinically observed time-course of thrombocytopenia.

Generation of functional platelets from canine induced pluripotent stem cells.

Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently, there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1, and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into MKs and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.

Three-stage ex vivo expansion of high-ploidy megakaryocytic cells: toward large-scale platelet production.

Hematopoietic stem and progenitor cells (HSPCs) have been cultured using a wide variety of cytokines to promote differentiation into megakaryocytic cells (Mks), the precursors to platelets. Greater Mk DNA content, or ploidy, has been correlated with increased platelet release. Gradients of pH, pO2, and signaling factors regulate megakaryopoiesis in the bone marrow niche. In this study, we demonstrate that a 3-phase culture process with increasing pH and pO2 and different cytokine cocktails greatly increases megakaryocyte production. CD34(+) HSPCs were first cultured at 5% O2 and pH 7.2 with a cytokine cocktail previously shown to promote Mk progenitor production. At day 5, cells were shifted to 20% O2 and pH 7.4 and maintained in 1 of 17 cytokine cocktails identified using a 2(4) factorial design of experiments method to evaluate the effects of interleukin (IL)-3, IL-6, IL-9, and high- or low-dose stem cell factor (SCF), in conjunction with thrombopoietin (Tpo) and IL-11, on expansion of mature Mks from progenitors. The combination of Tpo, high-dose SCF, IL-3, IL-9, and IL-11 best promoted Mk expansion. IL-3 greatly increased total cell fold expansion, but this was partially offset by lower Mk purity. IL-9 promoted CD41 and CD42b expression. High-dose (100 ng/mL) SCF increased Mk production and ploidy. Different commercial media and IL-3 sources substantially impacted differentiation, and X-VIVO 10 serum-free media best supported mature Mk expansion. Shifting from pH 7.4 to pH 7.6 at day 7 increased Mk production by 30%. Treatment with nicotinamide at day 7 or day 8 more than doubled the fraction of high-ploidy (>4N) Mks. Ultimately, the 3-phase culture system gave rise to 44.5±8.1 Mks and 8.5±3.1 high-ploidy Mks per input HSPC. Further optimization was required to improve platelet production. Using Iscove's modified Dulbecco's medium (IMDM)+20% BSA, insulin and transferin (BIT) 9500 Serum Substitute greatly improved the frequency and quality of Mk proplatelet extensions without affecting Mk expansion, commitment, or polyploidization in the 3-phase process. Mks cultured in IMDM+20% BIT 9500 gave rise to platelets with functional activity similar to that of fresh platelets from normal donors, as evidenced by basal tubulin distribution and the expression of surface markers and spreading in response to platelet agonists.

An herbal decoction of Radix astragali and Radix angelicae sinensis promotes hematopoiesis and thrombopoiesis.

ETHNOPHARMACOLOGICAL RELEVANCE: A decoction containing Radix angelicae sinensis and Radix astragali (Danggui Buxue Tang, DBT) has been used to raise the "Qi" and nourish the "Blood". However, its effects on haematopoiesis and particularly thrombopoiesis have not been studied. AIMS: This study aims to examine the effects of DBT on hematopoiesis and thrombopoiesis. MATERIALS AND METHODS: A myelosuppression mouse model was treated with DBT (10mg/kg/day). Peripheral blood cells from DBT and thrombopoietin-treated samples were counted on days 0, 7, 14 and 21. Then CFU assays were used to determine the effects of DBT on the megakaryocytic progenitor cells and other lineages. Last, analyses of annexin V, caspase-3, and mitochondrial membrane potential were conducted in megakaryocytic cell line M-07e. RESULTS: Morphological examination showed that DBT treatment significantly increased the recovery of the megakaryocytic series. DBT significantly enhanced the platelet recovery and CFU-MK formation in vivo. DBT significantly promoted CFU-MK and CFU-F formation. Last, we observed the antiapoptotic effects of DBT on M-07e cells. CONCLUSION: DBT might promote haematopoiesis and thrombopoiesis in the mouse model through (i) directly promoting the growth of megakaryocytes; (ii) indirectly promoting the growth of bone marrow stromal cells; (iii) inhibiting apoptosis of megakaryocytes.

Fusion of OTT to BSAC results in aberrant up-regulation of transcriptional activity.

OTT/RBM15-BSAC/MAL/MKL1/MRTF-A was identified as a fusion transcript generated by t(1;22)(p13;q13) in acute megakaryoblastic leukemia. Previous studies have shown that BSAC (basic, SAP, and coiled-coil domain) activates the promoters containing CArG boxes via interaction with serum response factor, and OTT (one twenty-two) negatively regulates the development of megakaryocytes and myeloid cells. However, the mechanism by which OTT-BSAC promotes leukemia is largely unknown. Here we show that OTT-BSAC, but not BSAC or OTT strongly activates several promoters containing a transcription factor Yin Yang 1-binding sequence. In addition, although BSAC predominantly localizes in the cytoplasm and its nuclear translocation is considered to be regulated by the Rho-actin signaling pathway, OTT-BSAC exclusively localizes in the nucleus. Moreover, OTT interacts with histone deacetylase 3, but this interaction is abolished in OTT-BSAC. Collectively, these functional and spatial changes of OTT and BSAC caused by the fusion might perturb their functions, culminating in the development of acute megakaryoblastic leukemia.

Metallothionein in human thrombocyte precursors, CD61+ megakaryocytes.

The in vitro biosynthesis of metallothionein (MT) was investigated in thrombocyte precursors (megakaryocytes) isolated from human cord blood. Biosynthesis and induction of MT in magnetic cell sorting-separated CD61(+) megakaryocytes was confirmed by immunohistochemical staining using monoclonal mouse anti-MT. The presence of MT was detected both in the nuclear and in the cytoplasmic area. Using RT-PCR, in vitro upregulation/induction of total MT transcripts was observed in CD61(+) cells at 48 h post-treatment with 100 micromol/L of zinc supplement. Seven isoform-specific mRNAs namely, MT-1A, MT-1B, MT-1E, MT-1G, MT-1H, MT-1X, and MT-2A were detected in the similar cell populations left untreated with zinc.

Mice rendered severely deficient in megakaryocytes through targeted gene deletion of the thrombopoietin receptor c-Mpl have a normal skeletal phenotype.

To explore whether a functional relationship exists between megakaryocytes and the cellular processes responsible for bone formation, we examined if Mpl ( -/- ) mice, which are severely megakaryocyte-deficient through c-Mpl gene deletion, have an abnormal skeletal phenotype compared to Mpl ( +/- ) and wild-type littermates. We also analyzed whether the osteogenic response to high-dose estrogen treatment is altered in Mpl ( -/- ) mice. Megakaryocyte numbers and skeletal indices were compared between Mpl ( -/- ) mice and littermate Mpl ( +/- ) and wild-type 12-week-old mice (six per group). Dual-energy X-ray absorbtiometry of whole body, excised tibias, and femurs was performed. Histomorphometric analyses of the proximal metaphysis and mid-diaphysis were carried out on longitudinal and transverse sections, respectively. Histomorphometry was performed on the proximal tibial metaphysis of four Mpl ( -/- ) and four wild-type mice following high-dose estrogen treatment (0.5 mg/animal/week) for 4 weeks. Mpl ( -/- ) mice had 10% the megakaryocyte number of Mpl ( +/- ) and wild-type littermates. Bone mineral density values in Mpl ( -/- ) mice were identical to those in Mpl ( +/- ) and wild-type mice for whole body, femur, and tibia. Histomorphometric analysis demonstrated that cancellous and cortical tibial bone parameters were similar across all genotypes. The osteogenic response to estrogen treatment was indistinguishable between Mpl ( -/- )and wild-type mice. We found that mice severely deficient in megakaryocytes have a normal skeletal phenotype. Additionally, the deficiency did not diminish the osteogenic marrow response to high-dose estrogen treatment. These results represent the first in vivo evidence that severe megakaryocyte deficiency does not affect bone formation, suggesting that this process is not dependent on normal megakaryocyte number.