OBJECTIVETo investigate the mRNA expression profiles of megakaryocytes (MKs) from human cord blood CD34+ cells ex vivo expanded using Solexa technique.

METHODSCD34+ Cells were isolated using density gradient centrifugation and magnetic activated cell sorting. Cultures were stimulated with recombinant human thrombopoietin (100 ng/ml). After 12 days, the MKs fraction was separated from the non-MKs fraction using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The mRNA expression of MKs and non-MKs was detected by Solexa sequencing.

RESULTSWe obtained 3 773 147 and 3 533 805 Tags from MKs and non-MKs, respectively. The amounts of unambiguous tags were 3 291 132 and 2 967 947 and those of distinct tags were 197 769 and 245 318. The expression of 1161 genes was up-regulated and that of 902 genes down-regulated. The expression of 2717 tags was up-regulated and that of 1519 tags down-regulated.

CONCLUSIONSMKs and non-MKs have remarkably different mRNA expression profiles. The differential gene-encoded products may be involved in cellular development, adhesion, apoptosis metabolism, intra- and intercellular signal transduction, and immune response. Further studies on this topic may clarify the expression mechanism, signal transduction, and regulation mechanisms.

Antigens, CD34 ; Cells, Cultured ; Fetal Blood ; cytology ; Humans ; Megakaryocytes ; cytology ; metabolism ; RNA, Messenger ; genetics ; Transcriptome

Objective: To establish an intramedullary transplantation model of primary megakaryocytes to evaluate the platelet-producing capacity of megakaryocytes and explore the underlying regulatory mechanisms. Methods: Donor megakaryocytes from GFP-transgenic mice bone marrow were enriched by magnetic beads. The platelet-producing model was established by intramedullary injection to recipient mice that underwent half-lethal dose irradiation 1 week in advance. Donor-derived megakaryocytes and platelets were detected by immunofluorescence staining and flow cytometry. Results: The proportion of megakaryocytes in the enriched sample for transplantation was 40 to 50 times higher than that in conventional bone marrow. After intramedullary transplantation, donor-derived megakaryocytes successfully implanted in the medullary cavity of the recipient and produce platelets, which showed similar expression of surface markers and morphology to recipient-derived platelets. Conclusion: We successfully established an in vivo platelet-producing model of primary megakaryocytes using magnetic-bead enrichment and intramedullary injection, which objectively reflects the platelet-producing capacity of megakaryocytes in the bone marrow.
Animals ; Blood Platelets ; Bone Marrow ; Bone Marrow Cells ; Bone Marrow Transplantation ; Humans ; Megakaryocytes/metabolism* ; Mice

5-hydroxtryptamine (5-HT, serotonin) has been recognized not only as a neurotransmitter and vasoactive agent, but also as a growth factor. 5-HT mainly binds to 5-HT(2) receptors or 5-HT(1) receptors on cell surface to stimulate cell proliferation through Ras or MAPK pathway in many cell types. It has been reported that 5-HT stimulates megakaryocytopoiesis via 5-HT receptors. The possible mechanism of 5-HT on the proliferation and differentiation of megakaryocytes (MK) has been discussed in this review article. In early stage of megakaryocytopoiesis, 5-HT may bind to 5-HT(2B) receptor on megakaryocytes, and promotes their proliferation and differentiation. In the late stage, 5-HT may involve in the platelet release procedure by inducing nitric oxide (NO) synthesis via 5-HT(2A) receptors. 5-HT can also antagonize the apoptotic effect induced by thrombospondin-1 (TSP-1) which is a platelet alpha granule protein and has synergic effect with platelet-derived growth factor (PDGF) to enhance megakaryocytes proliferation. Therefore, 5-HT is likely to be an important substance in the feedback regulation of thrombopoiesis. In this review the 5-HT and its receptors, 5-HT as cell growth factor, pathway of 5-HT stimulating cell proliferation and influance of 5-HT on MK-progenitor cells were summarized.
Humans ; Megakaryocytes ; physiology ; Receptors, Serotonin ; metabolism ; Receptors, Serotonin, 5-HT2 ; metabolism ; Serotonin ; metabolism ; pharmacology ; Thrombopoiesis ; physiology ; Thrombopoietin ; physiology

OBJECTIVE: To induce hematopoietic progenitor/stem cells of umbilical cord blood to differentiate into mature megakaryocytes and platelets in vitro and to investigate the mechanism of production of platelets. METHODS: The CD34+ cells were sorted from umbilical cord blood by magnetic activated cell sorting (MACS) and then cultured in vitro with optimized medium to be differentiated into mature megakaryocytes and platelets. The cultured cells and the platelet-like particles were isolated from the culture and were checked by the fluorescence-activated cell sorter (FACS), immunohistochemistry assays, light microscope,electron microscope and platelet aggregation tests. RESULTS: The cultured megakaryocytes were detected with proplatelets and both the cultured cells and the platelet-sized particles were found to have the same structure with the normal megakaryocytes and platelets by light and electron microscope. The immunohistochemistry assays revealed the cultured cells expressed GP II b III a with a positivity of 95% which was a special antigen for platelets and megakaryocytes. Culture-derived platelet-sized particles aggregated in response to thrombin as the plasma derived-platelets did. The cultured platelets had the same positivity of CD41 as the platelets from platelet rich plasma. CONCLUSION The hematopoietic progenitor/stem cells can be induced to differentiate into purified and mature megakaryocytes and platelets. It provides a practical way to study the mechanism of platelets production.
Antigens, CD34 ; metabolism ; Blood Platelets ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Fetal Blood ; cytology ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Megakaryocytes ; cytology

Platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic factor, is involved in the regulation of hematopoiesis and platelet production. Our studies demonstrate the presence of functional PDGF receptors (PDGFR) on human megakaryocytes/platelets and CD34(+) cells, and their ability to mediate a mitogenic response. PDGF promotes the ex vivo expansion of human hematopoietic stem (CD34(+)) and progenitor (CD41(+)) cells. More significantly, PDGF enhances the engraftment of human CD45(+) cells and their myeloid subsets (CD33(+), CD14(+) cells) in NOD/SCID mice. PDGF also stimulates in vitro megakaryocytopoiesis via PDGFR and/or the indirect effect on bone marrow microenvironment to produce TPO and other cytokines. It also shows a direct stimulatory effect of PDGF on c-Fos, GATA-1 and NF-E2 expressions in megakaryocytes. We speculate that these transcription factors may be involved in the signal transduction of PDGF on the regulation of megakaryocytopoiesis. PDGF also enhances platelet recovery in mouse model with radiation-induced thrombocytopenia. This radioprotective effect is likely to be mediated via PDGFR with subsequent activation of the PI3K/Akt pathway. It provides a possible explanation that blockage of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia.
Animals ; Hematopoietic Stem Cells ; cytology ; Humans ; Megakaryocytes ; cytology ; Mice ; Platelet-Derived Growth Factor ; metabolism ; Receptors, Platelet-Derived Growth Factor ; metabolism ; Thrombopoiesis

OBJECTIVEChemokine receptor CXCR4 and its ligand stromal-derived factor 1 alpha (SDF-1alpha) have been paid increasing attention for their involvement in megakaryocytic hematopoiesis. It has been revealed in recent years that they can induce mature and immature megakaryocytes (MKs) to migrate through bone marrow endothelial cells (BMEC) by increasing the affinity of MKs for BMEC. Thus MKs maturity and eventual release of platelet from MKs ensues. While maturity disturbance of MKs and impaired production of platelets have been regarded as the main pathogenesis of ITP, the mechanism of which still remains unclear. Therefore, a clear understanding of the levels of CXCR4 and SDF-1alpha within bone marrow in children with ITP will help us to elucidate further the mechanism of ITP as well as to provide direct theoretical evidence for predicting treatment effect and evaluating prognosis.

METHODSBone marrow were aspirated from 28 children with AITP and 12 normal children. Percoll density gradient and immunomagnetic beads method were used to purify megakaryocytes from the bone marrow. The immune cytochemistry was used to detect CXCR4 on megakaryocytes. The levels of SDF-1alpha were detected by ELISA. SPSS10.0 statistical software was used to deal with the experimental data.

RESULTSBefore the treatment in children with AITP, both the CXCR4 expression on megakaryocytes and the SDF-1alpha level in bone marrow plasma were markedly decreased compared with the normal controls (P < 0.05). As to the cases who were sensitive to the high-dose intravenous immunoglobulin (HDIVIgG), the CXCR4 and SDF-1alpha levels were much higher in children after the treatment than those before the treatment (P < 0.05). In 6 cases insensitive to HDIVIgG, before the treatment the CXCR4 level was much lower than the children sensitive to HDIVIgG (P < 0.05).

CONCLUSIONSThe low levels of CXCR4/SDF-1alpha system in bone marrow may be one of the factors which contribute to the maturity disturbance of megakaryocytes and disturbance of platelets production in AITP, while decreased CXCR4/SDF-1alpha system may be caused by the effect of autoantibody against platelet. The mechanism of HDIVIgG in the treatment of AITP may involve in the increasing expression of CXCR4/SDF-1alpha system. The level of CXCR4 on megakaryocytes may play a certain role in predicting the treatment effect of immunoglobulin.

Adolescent ; Bone Marrow ; metabolism ; Chemokine CXCL12 ; Chemokines, CXC ; blood ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Humans ; Infant ; Ligands ; Megakaryocytes ; metabolism ; Purpura, Thrombocytopenic, Idiopathic ; blood ; Receptors, CXCR4 ; biosynthesis

OBJECTIVEIn order to investigate whether or not thrombopoietin (TPO) could promote the fibrogenesis of bone marrow stromal cells in absence of megakaryocytes (MKs).

METHODSImproved dexter culture system with various TPO concentrations was used for ex vivo culture of bone marrow stromal cells. Relative proliferation index, the expressions of fibronectin, laminin and type IV collagen, and the systhesis of type III procollagen were detected at different time points during culture process.

RESULTSTPO stimulated the proliferation of bone marrow stromal cells. Relative proliferation index of the stromal cells increased with the TPO concentration increasing, and was not related to the exposure time. The expressions of fibronectin, laminin, and type IV collagen appeared stronger in the TPO groups than those in the control group. But the expressions of these molecules were not dependent upon the culture time. TPO could accelerate the synthesis of type III procollagen in bone marrow stromal cells, and this acceleration was unrelated to the TPO concentration.

CONCLUSIONThese findings suggested that TPO could stimulate the stromal cells with a consequence of increased syntheses and secretions of the extracellular matrix and collagen in absence of MKs. In other words, TPO could promote the fibrogenesis of bone marrow stromal cells without the existence of MKs.

Cells, Cultured ; Collagen Type III ; metabolism ; Collagen Type IV ; metabolism ; Extracellular Matrix ; metabolism ; Fibronectins ; metabolism ; Fibrosis ; pathology ; Humans ; Laminin ; metabolism ; Megakaryocytes ; cytology ; Mesenchymal Stromal Cells ; cytology ; metabolism ; pathology ; Thrombopoietin ; pharmacology

AIMIn order to investigate the effect of mesenchymal stem/progenitor cells on proliferation and differentiation towards megakaryocytes of CD34+ cells from human umbilical cord blood in vitro.

METHODSAfter mesenchymal stem/progenitor cells were advancely planted in DMEM medium and grown up to 80%, then the CD34+ cells were added to culture with mesenchymal stem/ progenitor cells or without mesenchymal stem/progenitor cells in DMEM for 14 days with TPO + IL-3 + SCF, TPO + IL-3 + SCF + IL-11 respectively. After cultured for 14 days, mononuclear cells (MNCs) were counted by automatic cell analyzer. The number of CD41+ cells and platelets were detected by flow cytometry. Platelets function were assessed through platelet aggregation test which was induced by thrombin.

RESULTSAs compared with the control group, the number of MNCs of co-culture system was not increased significantly (P > 0.05), but the number of CD4+ cells and platelets were increased significantly (P < 0.05). The platelets were aggregated by thrombin induced which could be seen in microscope or flow cytometry.

CONCLUSIONIt is concluded that mesenchymal stem/progenitor cells may be promoted to induce the cord blood CD34+ cells to differentiate towards megakaryocyte in the culture medium.

Antigens, CD34 ; metabolism ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Fetal Blood ; cytology ; Humans ; Megakaryocytes ; cytology ; Mesenchymal Stromal Cells ; cytology ; physiology

OBJECTIVETo analyze the ultra microstructures and the expression of platelet peroxidase (PPO) of megakaryocytes from bone marrow, their clinical manifestations and laboratory characteristics in patients with acute megakaryoblastic leukemia (AMKL).

METHODSKaryocytes from bone marrow of 22 AMKL patients were divided into two parts by lymphocyte separation liquid, one part was used to prepare the ordinary transmission electron microscope specimens to observe the morphological structures of megakaryocytes, the other was used to prepare the histochemical specimens of platelet peroxidase to analyze the positive reaction of PPO in AMKL, which were coupled with the patients' data of with bone marrow morphology, cell chemistry, and chromosome karyotype examination.

RESULTSMegakaryocytes from 17 of 22 patients were in the first stage, less than 20 µm in diameter, the nucleis were round, the cytoplasm contained microtubules, membranous vesicles and minute dense granules, no demarcation membrane system and surface-connected canalicular system, less dense granules and α-granules; Megakaryocytes in 5 cases were mainly in the first stage, while containing second and third stage megakaryocytes; the positive rate of PPO in megakaryocytes of 22 patients was 0-80%. The primitive and naive megakaryocytes were found in bone marrow smears of 22 cases, CD41 staining of the megakaryocytes was detected in the primitive and naive megakaryocytes, and more complex chromosome karyotype anomalies were observed.

CONCLUSIONThe majority of megakaryocytes in AMKL patients were the first stage ones, the rest were second and third stage ones, and the positive PPO reaction was significantly different. CD41 staining of the megakaryocytes was specific with complex chromosome karyotypeswere.

Blood Platelets ; enzymology ; Bone Marrow ; pathology ; Cell Count ; Chromosome Aberrations ; Chromosome Disorders ; Humans ; Karyotyping ; Leukemia, Megakaryoblastic, Acute ; diagnosis ; pathology ; Megakaryocytes ; pathology ; Peroxidase ; metabolism ; Staining and Labeling

To investigate the effect of costimulatory factors in the pathogenesis of chronic idiopathic thrombocytopenic purpura (CITP), we examined the expression of CD80 on platelets and megakaryocytes in patients with CITP and the controls by FACS. By using CD80 monoclonal antibody (McAb) to inhibit interaction among cells which is mediated by costimulatory factors, we observed the effect of CD80 McAb on the growth and maturation of megakaryocytic progenitors of patients with CITP in vitro. The results showed the expression of CD80 on platelets and megakaryocytes in CITP group was significantly higher than that in controls (P<0.01). There was a significantly positive correlation between the expression of CD80 on platelets and serum PAIgG in CITP (r=0.86, P<0.05). The mean of various clone numbers (CFU-MK, BFU-MK and mCFU-MK) in CITP were all lower than those in controls (P<0.05). In megakaryocytes co-cultured with CD80 McAb, there was an increasing tendency of the number of CFU-MK and big CFU-MK (the number of megakaryocyte with GP IIIa positive was more than 20) and mediate CFU-MK (the number of megakaryocyte with GP IIIa positive was 11-20). When the concentration of CD80 McAb was 10 microg/L, there was a significant difference in the number of megakaryocytic colony formation (CFU-MK, BFU-MK and mCFU-MK) between the group with CD80 McAb and that without it (P<0.05). These showed the abnormality of costimulatory factors had important effect in the pathogenesis of CITP.
Adult ; Antibodies, Monoclonal ; B7-1 Antigen ; biosynthesis ; Blood Platelets ; metabolism ; Cells, Cultured ; Chronic Disease ; Colony-Stimulating Factors ; metabolism ; Female ; Hematopoietic Stem Cells ; metabolism ; Humans ; Male ; Megakaryocytes ; cytology ; metabolism ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; etiology ; metabolism