Name: immunophenotyping and cell sorting of human mks from human primary sources or differentiated in vitro from hematopoietic progenitors
Uploaded: 2021-08-07T15:00:00
Description: Watch this Scientific Journal Video about Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors at JoVE.com

We thank Marcos P&#233;rez Basterrechea, Lorena Rodr&#237;guez Lorenzo and Bego&#241;a Garc&#237;a M&#233;ndez (HUCA) and Paloma Cerezo, Almudena Payero and Mar&#237;a 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 Econom&#237;a y Competitividad, Spain) and an I+D 2017 grant (SAF2017-85489-P; Ministerio de Ciencia, Innovaci&#243;n y Universidades, Spain and Fondos FEDER) to L.G., a Severo Ochoa Grant (PA-20-PF-BP19-014; Consejer&#237;a de Ciencia, Innovaci&#243;n y Universidades del Principado de Asturias, Spain) to P.M.-B. and an intramural postdoctoral grant 2018 (Fundaci&#243;n para la Investigaci&#243;n y la Innovaci&#243;n Biosanitaria de Asturias - FINBA, Oviedo, Spain) to A.A.-H. We thank Reinier van der Linden for sharing his knowledge (and time), especially his wise advice on multi-color tagged-antibody panel mix and single-color bead control preparation.

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 Cl&#237;nico San Carlos (HCSC).
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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...

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 different granules inherited by mature platelets1,2,3. As a result, mature MKs are large cells (40-60 &#181;m) characterized by a highly polyploid nucleus (reaching even &#62;64N). Recent studies suggest alternative routes by which HSCs differentiate into MKs bypassing traditional lineage commitment checkpoints in response to certain physio-pathological conditions4,5,6,7,8,9,10,11. These findings highlight that hematopoietic differentiation towards the mature MK is a continuum and adaptive process that responds to biological needs.
With the increasing knowledge on the cell biology and the molecular aspects characterizing megakaryopoiesis12, most of the research dedicated to the study of the process by flow cytometry are limited to the identification of mature MKs using lineage-specific surface markers (i.e., CD42A/B, CD41/CD61), while earlier MK differentiation stages remain unexplored. We previously documented a strategy to stage megakaryopoiesis in mouse bone marrow and bone marrow-derived MK cultures13,14, which we have adapted and applied to humans15. In the present article we show an immunophenotyping strategy that allows the characterization of megakaryopoiesis, from HSCs to mature MKs, in human primary sources (bone marrow -BM- and peripheral blood -PB-) or in vitro cultures using a panel integrating MK specific and non-specific surface markers (CD61, CD42B, CD49B, CD31, KIT and CD71, amongst others). Despite its large size and fragility, MKs can be immunophenotyped using the above-mentioned cell surface markers and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter to minimize cell rupture and/or damage. This technique facilitates multi-Omics approaches, with the aim to better understand the complexity of megakaryopoiesis and platelet production in human health and disease. Noteworthy, it will pose as a useful tool to aid diagnosis and prognosis in a clinical context of growing demand.
In this manuscript we document a strategy to stage human megakaryopoiesis with a panel integrating MK-specific and non-specific surface markers from primary sources or generated in vitro. Additionally, we provide a protocol to sort, with a fluorescence-activated cell sorter, the preferred fractions and mature MKs (Figure 1). This step is not popular, as it is technically difficult due to the large size and fragility of MKs. However, it has been employed both in mouse and human bone marrow samples previously, and due to technological advancement, with a better result each time16,17,18. Human primary sources where MKs or MK precursors can be studied include bone marrow, cord blood and peripheral blood, amongst other. The proper sample processing to isolate the relevant cell fraction for analysis on each sample is of importance. Standard procedures are incorporated, with some considerations to take into account when aiming at the study of megakaryopoiesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>130 micron Nozzle</td>
			<td>BD</td>
			<td>643943</td>
			<td>required for MK sorting</td>
		</tr>
		<tr>
			<td>5810R Centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Cell isolation and washes</td>
		</tr>
		<tr>
			<td>A-4-62 Swing Bucket Rotor</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Cell isolation and washes</td>
		</tr>
		<tr>
			<td>Aerospray Pro Hematology Slide Stainer / Cytocentrifuge</td>
			<td>ELITech Group</td>
			<td></td>
			<td>Automatized cytology devise, where slides are stained with Mat-Gr&#252;nwald Giemsa</td>
		</tr>
		<tr>
			<td>CO2 Incubator Galaxy 170 S</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Cell Incubation</td>
		</tr>
		<tr>
			<td>Cytospin 4 Cytocentrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>To prepare cytospins</td>
		</tr>
		<tr>
			<td>FACSAria IIu sorter</td>
			<td>BD</td>
			<td></td>
			<td>Lasers 488-nm and 633-nm</td>
		</tr>
		<tr>
			<td>FACSCanto II flow cytometer</td>
			<td>BD</td>
			<td></td>
			<td>Lasers 488-nm , 633-nm and 405-nm</td>
		</tr>
		<tr>
			<td>Olympus Microscope BX 41</td>
			<td>Olympus</td>
			<td></td>
			<td>Microphotographs</td>
		</tr>
		<tr>
			<td>Olympus Microscope BX 61</td>
			<td>Olympus</td>
			<td></td>
			<td>Microphotographs</td>
		</tr>
		<tr>
			<td>Zoe Fluorescent Cell Imager</td>
			<td>BioRad</td>
			<td></td>
			<td>Microphotographs</td>
		</tr>
		<tr>
			<td>To obtain PBMCs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipids Cholesterol Rich from adult bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>L4646</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stem Cell Technologies</td>
			<td>#07801</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Recombinant human Erythropoietin (EPO)</td>
			<td>&#160;R&#38;D Systems</td>
			<td>287-TC-500</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Recombinant human stem cell factor (SCF)</td>
			<td>Thermo Fisher Scientific, Gibco&#8482;</td>
			<td>PHC2115</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Recombinant human thrombopoietin (TPO)</td>
			<td>Thermo Fisher Scientific, Gibco&#8482;</td>
			<td>PHC9514</td>
			<td>or TPO receptor agonists</td>
		</tr>
		<tr>
			<td>StemSpan SFEM</td>
			<td>Stem Cell Technologies</td>
			<td>#09650</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Merck</td>
			<td>A7906-100G</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>BD CompBead Anti-Mouse Ig, &#954;/Negative Control Compensation Particles Set</td>
			<td>BD</td>
			<td>552843</td>
			<td>Antibodies for human cells are generally from mouse.</td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm</td>
			<td>BD</td>
			<td>554714</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>BD FACS Accudrop Beads</td>
			<td>BD</td>
			<td>345249</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 AF-647</td>
			<td>BD</td>
			<td>561654</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD31 FITC</td>
			<td>Immunostep</td>
			<td>31F-100T</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 FITC</td>
			<td>BD</td>
			<td>555821</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD41 PE</td>
			<td>BD</td>
			<td>555467</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD41 PerCP-Cy5.5</td>
			<td>BD</td>
			<td>333148</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD42A APC</td>
			<td>Immunostep</td>
			<td>42AA-100T</td>
			<td>We observed unspecific binding... that needs to be assessed</td>
		</tr>
		<tr>
			<td>CD42A PE</td>
			<td>BD</td>
			<td>558819</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD42B PerCP</td>
			<td>Biolegend</td>
			<td>303910</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD49B PE</td>
			<td>BD</td>
			<td>555669</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD61 FITC</td>
			<td>BD</td>
			<td>555753</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD71 APC-Cy7</td>
			<td>Biolegend</td>
			<td>334109</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BD Fc Block</td>
			<td>BD</td>
			<td>564219</td>
			<td>Fc blocking - control</td>
		</tr>
		<tr>
			<td>KIT PE-Cy7</td>
			<td>Biolegend</td>
			<td>313212</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>Lineage Cocktail 2 FITC</td>
			<td>BD</td>
			<td>643397</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>RNAse</td>
			<td>Merck</td>
			<td>R6513</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Triton X-500</td>
			<td>Merck</td>
			<td>93443-500ML</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Cell strainers for sorting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics Filters 100 micrometers</td>
			<td>Sysmex</td>
			<td>04-004-2328</td>
			<td>Cell strainers</td>
		</tr>
		<tr>
			<td colspan="4">Note: we do not specify general reagents/chemicals (PBS, EDTA, etc) or disposables (tubes, etc), or reagents specified in previous published and standard protocols&#160; - unless otherwise specified.</td>
		</tr>
	</tbody>
</table>

immunophenotyping and cell sorting of human mks from human primary sources or differentiated in vitro from hematopoietic progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). 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.

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+...

Watch this Scientific Journal Video about Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors at JoVE.com

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

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.

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

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

Hello.I am Andrea Acebes Huerta.Postdoctoral Researcher at the Platelet Research Lab part of the Health Research Institute of the Principality of Asturias, ISPA.Which is located in Oviedo, Spain.Hello.I am Laura Guti

Research

JoVE Journal

Biology

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...