Utilization of imaging flow cytometry to define intermediates of megakaryopoiesis in vivo and in vitro

McGrath, Kathleen E.

doi:10.1016/j.jim.2015.03.002

Cited by 8 publications

(5 citation statements)

References 17 publications

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“…In mouse studies, the use of reporter mice expressing a nuclear-localized fluorescent protein can help in discriminating MKs from cells aggregated with platelets. The use of imaging flow cytometry is another way to differentiate MKs from leukocyte–platelet aggregates ( 114 ). It also adds morphological information using the shape, density, texture, and coincidence of fluorescence to discriminate different MK maturation steps ( 115 ).…”

Section: Mk Identity and Methodology Advices For Further Studiesmentioning

confidence: 99%

Occurrence and role of lung megakaryocytes in infection and inflammation

2022

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Megakaryocytes (MKs) are large cells giving rise to platelets. It is well established that in adults, MKs develop from hematopoietic stem cells and reside in the bone marrow. MKs are also rare but normal constituents of the venous blood returning to the lungs, and MKs are found in the lung vasculature (MKcirc), suggesting that these cells are migrants from the bone marrow and get trapped in lung capillaries where the final steps of platelet production can occur. An unprecedented increase in the number of lung and circulating MKs was described in coronavirus disease 2019 (COVID-19) patients, suggesting that lung thrombopoiesis may be increased during lung infection and/or thromboinflammation. In addition to the population of platelet-producing intravascular MKs in the lung, a population of lung-resident megakaryocytes (MKL) has been identified and presents a specific immune signature compared to its bone marrow counterparts. Recent single-cell analysis and intravital imaging have helped us gain a better understanding of these populations in mouse and human. This review aims at summarizing the recent data on increased occurrence of lung MKs and discusses their origin, specificities, and potential role in homeostasis and inflammatory and infectious lung diseases. Here, we address remaining questions, controversies, and methodologic challenges for further studies of both MKcirc and MKL.

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Section: Mk Identity and Methodology Advices For Further Studiesmentioning

confidence: 99%

Occurrence and role of lung megakaryocytes in infection and inflammation

2022

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“…The exigency of separating mature MKs from their precursors is critical to define differentiation requirements and optimize ex vivo platelet production. Although megakaryocytes express specific surface markers facilitating their separation through flow cytometry [7], [12], [13], their rarity and susceptibility to shear stresses limit their isolation. Indeed, FACS, the gold-standard method for high-throughput cell sorting, loses efficacy when applied to cells with either large size, inherent fragility, and/or high buoyancy [14]- [16].…”

Section: Introductionmentioning

confidence: 99%

MarrowDLD: a microfluidic method for label-free retrieval of fragile bone marrow cells

Porro,

Sarkis,

Obergozo

et al. 2023

Preprint

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Functional bone marrow studies have focused primarily on hematopoietic progenitors, leaving limited knowledge about other fragile populations, such as bone marrow adipocytes (BMAds) and megakaryocytes. The isolation of these cells is challenging due to rupture susceptibility and large size. We introduce here a label-free cytometry microsystem, MarrowDLD, based on deterministic lateral displacement. MarrowDLD enables the isolation of fragile cells based on intrinsic size properties while preserving their viability and functionality. Bone marrow adipocytes, obtained from mouse and human stromal line differentiation, as well as megakaryocytes, from primary human CD34+ hematopoietic stem and progenitor cells, were used for validation. Precise micrometer-range separation cutoffs were adapted for each cell type. Cells were sorted directly in culture media, without pre-labeling steps, and with real-time imaging for quality control. At least 106 cells were retrieved intact per sorting round. Our method outperformed two FACS instruments in purity and yield, particularly for large cell size fractions. MarrowDLD represents a non-destructive sorting tool for fragile cells, facilitating the separation of phenotypically pure populations of BMAds and megakaryocytes to further investigate their physiological and pathological roles.

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“…The exigency of separating mature MKs from their precursors is critical to define differentiation requirements and optimize ex vivo platelet production. Although megakaryocytes express specific surface markers facilitating their separation through flow cytometry 7 , 11 , 12 , their rarity and susceptibility to shear stress forces limit their isolation. Indeed, FACS, the gold-standard method for high-throughput cell sorting, loses efficacy when applied to cells with either large size, inherent fragility, and/or high buoyancy 13 – 15 .…”

Section: Introductionmentioning

confidence: 99%

MarrowCellDLD: a microfluidic method for label-free retrieval of fragile bone marrow-derived cells

Porro,

Sarkis,

Obergozo

et al. 2023

Sci Rep

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Functional bone marrow studies have focused primarily on hematopoietic progenitors, leaving limited knowledge about other fragile populations, such as bone marrow adipocytes (BMAds) and megakaryocytes. The isolation of these cells is challenging due to rupture susceptibility and large size. We introduce here a label-free cytometry microsystem, MarrowCellDLD, based on deterministic lateral displacement. MarrowCellDLD enables the isolation of large, fragile BM-derived cells based on intrinsic size properties while preserving their viability and functionality. Bone marrow adipocytes, obtained from mouse and human stromal line differentiation, as well as megakaryocytes, from primary human CD34+ hematopoietic stem and progenitor cells, were used for validation. Precise micrometer-range separation cutoffs were adapted for each cell type. Cells were sorted directly in culture media, without pre-labeling steps, and with real-time imaging for quality control. At least 106 cells were retrieved intact per sorting round. Our method outperformed two FACS instruments in purity and yield, particularly for large cell size fractions. MarrowCellDLD represents a non-destructive sorting tool for large, fragile BM-derived cells, facilitating the separation of pure populations of BMAds and megakaryocytes to further investigate their physiological and pathological roles.

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Utilization of imaging flow cytometry to define intermediates of megakaryopoiesis in vivo and in vitro

Cited by 8 publications

References 17 publications

Occurrence and role of lung megakaryocytes in infection and inflammation

Occurrence and role of lung megakaryocytes in infection and inflammation

MarrowDLD: a microfluidic method for label-free retrieval of fragile bone marrow cells

MarrowCellDLD: a microfluidic method for label-free retrieval of fragile bone marrow-derived cells

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