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Biology

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Published: August 7th, 2021

DOI:

10.3791/62569

1Platelet Research Lab, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), 2Flow Cytometry and Cell Sorting Platform (ISPA), 3Clinical Diagnosis Laboratory - Dept. of Hematology, Hospital Universitario Central de Asturias (HUCA), 4Department of Hematology, Hospital Universitario Severo Ochoa (HUSO), 5Department of Hematology, Instituto de Investigación Sanitaria San Carlos (IdISSC), Hospital Clínico San Carlos (HCSC), 6Dept. of Medicine, University of Oviedo

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even >64N) and extremely large cell (40-60 µm). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Megakaryocytes (MKs) develop from hematopoietic stem cells (HSCs) following a complex process called megakaryopoiesis, which is orchestrated mainly by the hormone thrombopoietin (TPO). The classical view of megakaryopoiesis describes the cellular journey from HSCs through a succession of hierarchical stages of committed progenitors and precursor cells, leading ultimately to a mature MK. During maturation, MKs experience multiple rounds of endomitosis, develop an intricate intracellular demarcation membrane system (DMS), which provides enough membrane surface for platelet production, and efficiently produce and pack the plethora of factors that are contained in the dif....

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Whole blood and bone marrow samples were obtained and processed in accordance with the 1964 Declaration of Helsinki. Whole blood samples were obtained from healthy donors after giving informed consent (ISPA), within a study approved by our institutional medical ethical committee (Hospital Universitario Central de Asturias -HUCA-). Bone marrow samples were obtained from bone marrow aspirate discard material of patients managed at the Dept. of Hematology of the Hospital Clínico San Carlos (HCSC).

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Bone Marrow and Ploidy
In Figure 4, we show a representative immunophenotyping analysis of megakaryopoiesis in BM samples (aspiration) from patients. When plotting the cellular fraction against CD71 and CD31, we have gated six main populations: CD31- CD71- (red), CD31- CD71+ (blue), CD31+ CD71- (orange), CD31+ CD71mid (light green), CD31+ CD71+.......

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Most of the research focusing on the study of megakaryopoiesis by flow cytometry is to date limited to the identification of MK subsets using only lineage-specific surface markers (i.e., CD42A/CD42B, CD41/CD61), while earlier MK differentiation stages have been poorly examined. In the present article we show an immunophenotyping strategy to address a comprehensive flow cytometry characterization of human megakaryopoiesis. Overall, we would like to highlight the utility of combining MK specific and non-specific s.......

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We thank Marcos Pérez Basterrechea, Lorena Rodríguez Lorenzo and Begoña García Méndez (HUCA) and Paloma Cerezo, Almudena Payero and María de la Poveda-Colomo (HCSC) for technical support. This work was partially supported by Medical Grants (Roche SP200221001) to A.B., an RYC fellowship (RYC-2013-12587; Ministerio de Economía y Competitividad, Spain) and an I+D 2017 grant (SAF2017-85489-P; Ministerio de Ciencia, Innovación y Universidades, Spain and Fondos FEDER) to L.G., a Severo Ochoa Grant (PA-20-PF-BP19-014; Consejería de Ciencia, Innovación y Universidades del Principado de Asturias, Spain) to P.M.-B. and an intram....

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Name Company Catalog Number Comments
130 micron Nozzle BD 643943 required for MK sorting
5810R Centrifuge Eppendorf Cell isolation and washes
A-4-62 Swing Bucket Rotor Eppendorf Cell isolation and washes
Aerospray Pro Hematology Slide Stainer / Cytocentrifuge ELITech Group Automatized cytology devise, where slides are stained with Mat-Grünwald Giemsa
CO2 Incubator Galaxy 170 S Eppendorf Cell Incubation
Cytospin 4 Cytocentrifuge Thermo Scientific To prepare cytospins
FACSAria IIu sorter BD Lasers 488-nm and 633-nm
FACSCanto II flow cytometer BD Lasers 488-nm , 633-nm and 405-nm
Olympus Microscope BX 41 Olympus Microphotographs
Olympus Microscope BX 61 Olympus Microphotographs
Zoe Fluorescent Cell Imager BioRad Microphotographs
To obtain PBMCs
Lipids Cholesterol Rich from adult bovine serum Sigma-Aldrich L4646 or similar
Lymphoprep Stem Cell Technologies #07801 or similar
Penicillin-Streptomycin Sigma-Aldrich P4333 or similar
Recombinant human Erythropoietin (EPO)  R&D Systems 287-TC-500 or similar
Recombinant human stem cell factor (SCF) Thermo Fisher Scientific, Gibco™ PHC2115 or similar
Recombinant human thrombopoietin (TPO) Thermo Fisher Scientific, Gibco™ PHC9514 or TPO receptor agonists
StemSpan SFEM Stem Cell Technologies #09650
Flow Cytometry Analyses
Bovine Serum Albumin Merck A7906-100G or similar
BD CompBead Anti-Mouse Ig, κ/Negative Control Compensation Particles Set BD 552843 Antibodies for human cells are generally from mouse.
BD Cytofix/Cytoperm BD 554714 or similar
BD FACS Accudrop Beads BD 345249
CD31 AF-647 BD 561654 Mouse anti-human
CD31 FITC Immunostep 31F-100T
CD34 FITC BD 555821 Mouse anti-human
CD41 PE BD 555467 Mouse anti-human
CD41 PerCP-Cy5.5 BD 333148 Mouse anti-human
CD42A APC Immunostep 42AA-100T We observed unspecific binding... that needs to be assessed
CD42A PE BD 558819 Mouse anti-human
CD42B PerCP Biolegend 303910 Mouse anti-human
CD49B PE BD 555669 Mouse anti-human
CD61 FITC BD 555753 Mouse anti-human
CD71 APC-Cy7 Biolegend 334109 Mouse anti-human
Hoechst 33342 Thermo Fisher Scientific H3570
Human BD Fc Block BD 564219 Fc blocking - control
KIT PE-Cy7 Biolegend 313212 Mouse anti-human
Lineage Cocktail 2 FITC BD 643397 Mouse anti-human
RNAse Merck R6513 or similar
Triton X-500 Merck 93443-500ML or similar
Cell strainers for sorting
CellTrics Filters 100 micrometers Sysmex 04-004-2328 Cell strainers
Note: we do not specify general reagents/chemicals (PBS, EDTA, etc) or disposables (tubes, etc), or reagents specified in previous published and standard protocols  - unless otherwise specified.

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