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

OBJECTIVETo 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.

METHODSBone 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.

RESULTSIn 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.

CONCLUSIONPDS-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.

Blotting, Western ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Colony-Forming Units Assay ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Gene Expression Profiling ; Ginsenosides ; pharmacology ; Humans ; Megakaryocytes ; cytology ; drug effects ; metabolism ; Patents as Topic ; Saponins ; pharmacology ; Stem Cells ; cytology ; drug effects ; Transcription Factors ; metabolism ; Up-Regulation ; drug effects ; genetics

OBJECTIVETo study the role of PDGF/PDGFR in essential thrombocythemia (ET) by investigating the expression of PDGF-BB in bone marrow and the expression of PDGFR-β in bone marrow cells of patients with ET and explore the new target for treating ET patients through inhibiting the PDGFR of megakaryocytes.

METHODSThe expression level of PDGF-BB in bone marrow of ET patients and normal controls were assayed by using ELISA, the expression level of PDGFR-β (CD140) in bone marrow of ET patients and normal controls were detected by using flow cytometry, the effect of PDGF-BB in JAK2/STAT3 and PI3K/AKT pathway was detected by using flow cytometry or Werstern blot, and the effect of imatinib on the megakaryopoiesis of PDGF was observed.

RESULTSThe expression level of PDGF-BB in bone marrow of ET patients was significantly higher than that in normal controls; the expression level of PDGFR-β in bone marrow of ET patients was significantly higher than that in nornal controls; PDGF-BB could activate JAK2/STAT3 and PI3K/AKT pathway of megakaryocytes, while the imatinib could block the effect of PDGF-BB on megakaryocyte.

CONCLUSIONThe elevated PDGF-BB and PDGFR-β may be involved in ET, and the physiopathologic mechanism is that the elevated PDGF-BB activates PDGFR with subsequent activation of the JAK2/STAT3 and PI3K/AKT pathways, stimulating megakaryopoiesis. Imatinib may have a therapeutical effect on ET via blocking of PDGFR.

Bone Marrow ; metabolism ; Case-Control Studies ; Humans ; Megakaryocytes ; metabolism ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-sis ; metabolism ; Receptor, Platelet-Derived Growth Factor beta ; metabolism ; Signal Transduction ; Thrombocythemia, Essential ; metabolism ; Thrombopoiesis

OBJECTIVETo investigate the effect and mechanism of Factor Xa on the differentiation of Meg-01 cells into platelet-like particles.

METHODSThe Meg-01 cells were used as experimental object, Factor Xa was used as agonist. Cell proliferation was detected by CCK-8 assay. The viability of platelet-like particles was analyzed by AlamaBlue kit. MAPK/ERK pathway and PI3K/AKT pathway were assayed by Western blot. The expression of CD41b was analyzed by Western blot and flow cytometry. Cell cycle and apoptosis were detected by flow cytometry.

RESULTSThe Factor Xa (1 µg/ml) inhibited cell viability, induced apoptosis. Factor Xa triggered cell arrest at the G(2)/M stage and down-regulated the expression of SKP2. After Meg-01 cells were stimulated by Factor Xa, the expression of CD41b was up-regulated and the MAPK/ERK pathway and PI3K/AKT pathway were activated. The platelets-like particles stimulated by FXa activation were viable.

CONCLUSIONThe Factor Xa maybe display some effect on the differentiation of megakaryocytes into platelets.

Apoptosis ; Blood Platelets ; cytology ; drug effects ; Cell Cycle Checkpoints ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; Cell Survival ; Factor Xa ; pharmacology ; Humans ; MAP Kinase Signaling System ; Megakaryocytes ; cytology ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism

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

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

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

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

In order to investigate the effect of stromal cell derived factor-1 (SDF-1)/CXCR4 on the proliferation of megakaryocytic line-HEL cells co-cultured with human umbilical cord blood-derived stromal cells (hUCBSCs) and to further elucidate the mechanism of SDF-1/CXCR4-mediated functions, the HEL cells were co-cultured with hUCBSCs or human bone marrow stromal cells (hBMSCs), the suspended HEL was used as control. The concentrations of SDF-1 in supernatant of hUCBSCs and hBMSCs were detected by ELISA assay. The expression of CXCR4 membrane-bound protein of HEL cells was detected by laser confocal scanning microscopy and flow cytometry, and the expression of CXCR4 mRNA was detected by RT-PCR. The result showed that the concentrations of SDF-1 in different groups were the same at the early stage of culturing. But at 6 days after seeding, the concentrations of SDF-1 increased significantly in the hUCBSCs group, even though the passage was done. By means of laser confocal microscopy, the expression of CXCR4 protein and also red dots of fluorescence could be detected in cytoplasm of HEL cells co-cultured with hUCBSCs. However, there was no significant differences of the CXCR4 mRNA level between different groups (p > 0.05). It is concluded that hUCBSCs may play important roles in secreting high level of SDF-1 and regulating megakaryocyte expression of CXCR4.
Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12 ; genetics ; metabolism ; Coculture Techniques ; Fetal Blood ; cytology ; metabolism ; Flow Cytometry ; Humans ; Megakaryocytes ; cytology ; metabolism ; Monocytes ; cytology ; RNA, Messenger ; genetics ; Receptors, CXCR4 ; genetics ; metabolism ; Stromal Cells ; cytology

OBJECTIVETo investigate the effect of Angelica polysaccharide (APS), platelet-derived growth factor (PDGF) and thrombopoietin (TPO) on the proliferation and apoptosis of human megakaryocytic cell line M-07e.

METHODSCell count and the viability testing of M-07e cells (trypan blue exclusion assay) were performed at 24 hours, 48 hours and 72 hours after treatment with APS, PDGF or TPO. Three apoptosis related flow cytometric assays including Annexin V, Caspase-3 and JC-1 were performed to determine apoptotic rate of each group at 72 hours after the treatment.

RESULTSAfter the incubation, the number of M-07e cells in the APS, PDGF and TPO group increased and the viabilities of the three groups were significantly higher than the control group (P < 0.05). The dead cells in the APS, PDGF and TPO group were (19.41 +/- 7.59)%, (21.38 +/- 7.25)% and (18.77 +/- 8.00)%, respectively by flow cytometry using Annexin V method, which were significantly lower compared to the control group (34.33 +/- 5.46)%. The expression of the activated caspase-3 in the group of APS, PDGF and TPO were (12.27 +/- 5.18)%, (12.39 +/- 6.26)% and (13.75 +/- 8.25)%, the APS and PDGF group decreased significantly compared to the control group (18.92 +/- 6.09)%. The ratio of total cell deaths in the APS, PDGF and TPO group were (23.64 +/- 6.69)%, (28.00 +/- 10.05)% and (27.99 +/- 8.99)%, the ratio in APS group decreased significantly compared to the control group (39.48 +/- 11.86)% by JC-1 method. Differences between APS and PDGF groups and between APS and TPO groups were not statistically significant.

CONCLUSIONAPS, PDGF and TPO have similar effect in stimulating proliferation and inhibiting serum-free-culture induced apoptosis of M-07e cells.

Angelica ; chemistry ; Apoptosis ; drug effects ; Benzimidazoles ; pharmacology ; Carbocyanines ; pharmacology ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Flow Cytometry ; Fluorescent Dyes ; pharmacology ; Humans ; Megakaryocytes ; drug effects ; physiology ; Organic Chemicals ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Thrombopoiesis ; Thrombopoietin ; pharmacology