Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions

Jackson, CW; Brown, LK; Somerville, BC; Lyles, SA; Look, A. Thomas

doi:10.1182/blood.v63.4.768.bloodjournal634768

Cited by 19 publications

(12 citation statements)

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“…The maturing MK population can be further characterized by distinguishing exhausted MKs, high ploidy nuclei with a thin rim of cytoplasm that remain following platelet production . Exhausted MKs are rarely studied as low levels of surface marker expression make them difficult to discriminate by standard FC; however, the morphologic analysis capacities afforded by IFC allow for the identification of this population . Subtracting the DRAQ5‐defined nuclear mask from the CD41‐based cell mask with Boolean logic creates a “cytoplasm” mask.…”

Section: Resultsmentioning

confidence: 99%

Improved quantitative analysis of primary bone marrow megakaryocytes utilizing imaging flow cytometry

et al. 2014

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Life-threatening thrombocytopenia can develop following bone marrow injury due to decreased platelet production from megakaryocytes (MKs). However, the study of primary MKs has been complicated by their low frequency in the bone marrow and by technical challenges presented by their unique maturation properties. More accurate and efficient methods for the analysis of in vivo MKs are needed to enhance our understanding of megakaryopoiesis and ultimately develop new therapeutic strategies for thrombocytopenia. Imaging flow cytometry (IFC) combines the morphometric capabilities of microscopy with the high-throughput analyses of flow cytometry (FC). Here, we investigate the application of IFC on the ImageStreamX platform to the analysis of primary MKs isolated from murine bone marrow. Our data highlight and address technical challenges for conventional FC posed by the wide range of cellular size within the MK lineage as well as the shared surface phenotype with abundant platelet progeny. We further demonstrate that IFC can be used to reproducibly and efficiently quantify the frequency of primary murine MKs in the marrow, both at steady-state and in the setting of radiation-induced bone marrow injury, as well as assess their ploidy distribution. The ability to accurately analyze the full spectrum of maturing MKs in the bone marrow now allows for many possible applications of IFC to enhance our understanding of megakaryopoiesis and platelet production.

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Section: Resultsmentioning

confidence: 99%

Improved quantitative analysis of primary bone marrow megakaryocytes utilizing imaging flow cytometry

et al. 2014

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“…5b). However, we reasoned that these results could be due to increases in cell size as it has been shown that the mean cell volume of MKs doubles with each ploidy doubling (Penington and Olsen, 1970; Odell et al, 1976; Levine et al, 1982; Jackson et al, 1984; Worthington et al, 1984; Tomer et al, 1988). Therefore, we compared the cyclin B level (relative MFI) of each G2/M population to the mean forward scatter (FSC) of that population and to ploidy (Fig.…”

Section: Resultsmentioning

confidence: 99%

Kinetics of endomitosis in primary murine megakaryocytes

Carow

Fox

Kaushansky

2001

Journal Cellular Physiology

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Megakaryocytes (MKs) develop from diploid progenitor cells via successive rounds of DNA synthesis in the absence of cell division, a process termed endomitosis (EnM). While the mechanism underlying EnM is not known, studies in yeast and leukemic cell lines have suggested that it may be due to reduced levels of cyclin B1 or cdc2, leading to a decrease in mitotic kinase activity. Using flow cytometry to study EnM highly purified marrow-derived MK precursors, we found that: (1) on average, 36% of 8N-32N MKs expressed abundant cyclin B during G2/M. The percentage of cells in G2/M decreased in >64N MKs, suggesting the limit of EnM, (2) the level of cyclin B per G2/M MK increased linearly with ploidy, (3) cyclin B expression oscillated normally in polyploid MKs, (4) MPM-2, a phosphoepitope created by the action of mitotic kinases and specific to M-phase cells, was expressed in a significant fraction of polyploid MKs, and (5) there was an apparent increase of cyclin B in G1-phase in polyploid MKs. This study provides the first qualitative kinetic data regarding the cell cycle status of MKs within individual ploidy classes. It also demonstrates the feasibility of using anti-cyclin B antibody and flow cytometry to resolve G1 from G2/M populations in polyploid MKs. Finally, these findings establish that neither a relative nor absolute deficiency of mitotic kinase components is responsible for EnM, suggesting that the departure from normal cell division kinetics seen in polyploid MKs is likely due to alterations in other cell cycle regulators.

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“…Aster subsequently corroborated this concept in humans who had undergone splenectomy (Aster, , ). Megakaryocyte size and ploidy are inversely related to the platelet count . Using a variety of techniques to measure megakaryocyte size and ploidy in animals, several studies showed that megakaryocyte size and ploidy increased as the platelet count fell, and both fell when the platelet count was experimentally increased by transfusion (Harker & Finch, ; Harker, ; Penington & Olsen, ; Jackson et al , ). Later studies showed that there was an inverse, log‐linear relationship between the platelet count and megakaryocyte DNA ploidy (Kuter & Rosenberg, ; Kuter, ).…”

Section: Physiological Observations Demonstrate That Platelet Productmentioning

confidence: 99%

Milestones in understanding platelet production: a historical overview

Kuter

2014

Br J Haematol

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SummaryThe discovery of thrombopoietin (TPO, also termed THPO) in 1994 was a major achievement in understanding the regulation of platelet production. In prior decades, physiological studies had demonstrated that platelets were produced from bone marrow megakaryocytes and that the megakaryocytes responded to thrombocytopenia by increasing their number, size and DNA ploidy. In 1958, it was proposed that a 'thrombopoietin' must exist that regulated this interaction between the circulating platelet mass and the bone marrow megakaryocytes. After over three decades of effort, TPO was finally purified by five independent laboratories. TPO stimulated megakaryocyte colony-forming cell growth and increased the number, size and ploidy of megakaryocytes. When the genes for TPO or TPO receptor were eliminated in mice, megakaryocytes grew and platelets were made, but at 15% of their normal number. A first generation of recombinant human (rh) TPO molecules [rhTPO and pegylated recombinant human megakaryocyte growth and development factor (PEG-rhMGDF)] rapidly entered clinical trials in 1995 and increased platelet counts in humans undergoing nonmyeloablative chemotherapy but not in those undergoing stem cell transplantation. Antibodies developed against PEGrhMGDF and development of these recombinant thrombopoietins ended. A second generation of TPO receptor agonists (romiplostim and eltrombopag) was then developed. Neither of these TPO receptor agonists demonstrated any significant untoward effects and both are now licensed in many countries for the treatment of immune thrombocytopenia. This review describes the significant experiments that have surrounded the discovery of TPO and its clinical development.

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Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions

Cited by 19 publications

References 0 publications

Improved quantitative analysis of primary bone marrow megakaryocytes utilizing imaging flow cytometry

Improved quantitative analysis of primary bone marrow megakaryocytes utilizing imaging flow cytometry

Kinetics of endomitosis in primary murine megakaryocytes

Milestones in understanding platelet production: a historical overview

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