Immunocytochemistry and flow cytometry evaluation of human megakaryocytes in fresh samples and cultures of CD34+ cells

Qiao, Xiaoying; Loudovaris, Maureen; Unverzagt, Kristen; Walker, Donald E.; Smith, Stephen L.; Martinson, Jeffrey; Schilling, Marta; Lee, Wanda; Williams, Stephanie; Epps, Dennis E. Van; Cohen, Isaac; Bender, James

doi:10.1002/(sici)1097-0320(19960301)23:3<250::aid-cyto8>3.0.co;2-m

Cited by 26 publications

(15 citation statements)

References 26 publications

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“…Megakaryocytes are also positive for CD41a and CD61, and measuring expression of these antigens allows quantitation of megakaryocytes within this gate. 16 Flow cytometry detected increased numbers of megakaryocytes (mean 24.6%, range 1%-76%) in MDS specimens compared with aplastic anemia specimens (mean 5.3%) but not compared with normals (mean 23%) or the remission bone marrows from patients with nonmyeloid neoplasia (mean 14%). Increased numbers of megakaryocytes were defined as more than 15% of the gate, which is 4 SDs above that observed in aplastic anemia patients.…”

Section: Flow Cytometry In Mds 983mentioning

confidence: 95%

Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome

Stetler‐Stevenson

Arthur

Jabbour

et al. 2001

Blood

269

260

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Section: Flow Cytometry In Mds 983mentioning

confidence: 95%

Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome

Stetler‐Stevenson

Arthur

Jabbour

et al. 2001

Blood

269

260

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“…To identify MKs in the bone marrow or from the spleen, surface expression of the thrombopoietic marker CD41 (integrin subunit: ␣IIb) was determined on cells with a high forward scatter. 24,25 CD41 ϩ MKs were then stained for the surface expression of CXCR4. Other samples were analyzed to determine the ploidy status of MKs using a TOPRO-3 DNA stain (Invitrogen Life Technologies).…”

Section: Flow Cytometric Analysis Of Mk Cxcr4 Expression and Determinmentioning

confidence: 99%

VEGFR1 stimulates a CXCR4-dependent translocation of megakaryocytes to the vascular niche, enhancing platelet production in mice

Pitchford

Lodie²,

Rankin

2012

Blood

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It has previously been reported that VEGF-A stimulates megakaryocyte (MK) maturation in vitro. Here we show that treatment of mice with the isoform VEGF-A 165 resulted in a significant increase in circulating numbers of platelets. Using specific VEGFR1 and VEGFR2 blocking mAbs and selective VEGFR1 and 2 agonists, PlGF-2 and VEGF-E, respectively, we show directly that stimulation of VEGFR1, but not VEGFR2, increases circulating platelet numbers in vivo. Using flow cytometric analysis of harvested MKs, we show that while PlGF does not change the absolute numbers of MKs present in the bone marrow and the spleen, it increases both their maturation and cell-surface expression of CXCR4 in the bone marrow. Histology of the bone marrow revealed a redistribution of MKs from the endosteal to the vascular niche in response to both VEGF-A 165 and PlGF-2 treatment in vivo. Antagonism of CXCR4 suppressed both the VEGFR1-stimulated redistribution of megakyocytes within the bone marrow compartment and the VEGF-A 165 -induced thrombocytosis. In conclusion, we define a novel proinflammatory VEGFR1-mediated pathway that stimulates the maturation and upregulation of CXCR4 on megakaryocytes, leading to their redistribution within the bone marrow environment, thereby enhancing platelet production in vivo. (Blood. 2012;120(14):2787-2795) IntroductionUnder homeostatic conditions, megakaryopoiesis is driven primarily by thrombopoietin (TPO) and stem cell factor (SCF). 1,2 Patients suffering from congenital amegakaryocytic thrombocytopenia (a mutation to the TPO receptor: cMPL), and mice with genetic deletions of SCF and TPO, therefore exhibit a profound thrombocytopenia. 3,4 Within the bone marrow endosteal niche, TPO acts synergistically with other hematopoietic cytokines to stimulate stem cell differentiation toward the megakaryocyte lineage to create promegakaryoblasts. 5 Promegakaryoblasts then begin to increase their ploidy content via the process of endomitosis. This process driven by TPO is essential for normal maturation into megakaryocytes (MKs). 6,7 Mature MKs then migrate to within close proximity of the bone marrow sinusoidal endothelium to form preplatelet buds, eventually releasing platelets. 8,9 Studies of megakaryopoiesis in vitro using CD34 ϩ cell unilineage differentiation cultures have shown that the upregulation of the chemokine receptor CXCR4 is associated with the maturation of megakaryocytes. 10 Although CXCL12 (the ligand for CXCR4) acting alone does not stimulate MK development, it has been reported to act synergistically with TPO to enhance megakaryopoiesis and platelet formation in vitro and in vivo. [11][12][13] The original studies describing the expression of CXCR4 on MKs demonstrated that CXCL12 stimulated their migration in vitro. 8,14 More recent studies have shown that adenoviral delivery of CXCL12, resulting in a sustained elevation in plasma CXCL12 over 10 days, increased circulating numbers of platelets in TPO-and MPL-deficient mice. It was shown that platelet production was enhanced, as a result o...

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“…Megakaryocyte/erythroid progenitor cells have an immunophenotype of Lin − CD34 + CD33 + D38 + IL3Rα−CD45RA− [24]. During differentiation into mature megakaryocytes, the expression of CD33 and CD34 on the cell surface decreases, while the expression of CD41a, CD42b, CD45, and CDw32 increases [23][24][25]. CD41a is a characteristic molecular marker of megakaryocyte differentiation and maturation [18].…”

Section: Discussionmentioning

confidence: 99%

The effect of amifostine on differentiation of the human megakaryoblastic Dami cell line

Wang

Yang

et al. 2016

Cancer Medicine

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Amifostine is a cytoprotective drug that was initially used to control and treat nuclear radiation injury and is currently used to provide organ protection in cancer patients receiving chemotherapy. Clinical studies have also found that amifostine has some efficacy in the treatment of cytopenia caused by conditions such as myelodysplastic syndrome and immune thrombocytopenia, both of which involve megakaryocyte maturation defects. We hypothesized that amifostine induced the differentiation of megakaryocytes and investigated this by exposing the human Dami megakaryocyte leukemia cell line to amifostine (1 mmol/L). After 12 days of amifostine exposure, optical microscopy showed that the proportion of Dami cells with diameters >20 μm had increased to 24.63%. Transmission electron microscopy identified the development of a platelet demarcation membrane system, while flow cytometry detected increased CD41a expression and decreased CD33 expression on the Dami cell surface. Ploidy analysis found that the number of polyploid cells with >4N DNA content increased to 27.96%. We did not detect any elevation in the mRNA or protein levels of megakaryocytic differentiation-associated transcription factors GATA-binding factor 1 (GATA-1) and nuclear factor, erythroid 2 (NF-E2), but nuclear import assay revealed an increased nuclear translocation of these proteins. These findings indicate that amifostine induced the differentiation of Dami cells into mature megakaryocytes via a mechanism involving increased nuclear translocation of the transcription factors, NF-E2 and GATA-1. Cancer Medicine Open Access 2013

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Immunocytochemistry and flow cytometry evaluation of human megakaryocytes in fresh samples and cultures of CD34+ cells

Cited by 26 publications

References 26 publications

Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome

Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome

VEGFR1 stimulates a CXCR4-dependent translocation of megakaryocytes to the vascular niche, enhancing platelet production in mice

The effect of amifostine on differentiation of the human megakaryoblastic Dami cell line

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