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.

<ol><li>Italiano, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unraveling+Mechanisms+That+Control+Platelet+Production%5BTitle%5D)+AND+%22Semin+Thrombosis+And+Haemostasis%22%5BJournal%5D)">Unraveling Mechanisms That Control Platelet Production.</a> Semin Thrombosis And Haemostasis. 39 (1), 15-24 (2013).</li>
<li>Machlus, K. R., Italiano, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Incredible+Journey%3A+From+Megakaryocyte+Development+To+Platelet+Formation%5BTitle%5D)+AND+%22Journal+Of+Cell+Biology%22%5BJournal%5D)">The Incredible Journey: From Megakaryocyte Development To Platelet Formation.</a> Journal Of Cell Biology. 201 (6), 785-796 (2013).</li>
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<li>Couldwell, G., Machlus, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+Of+Megakaryopoiesis+And+Platelet+Production+During+Inflammation%5BTitle%5D)+AND+%22Thrombosis+Research%22%5BJournal%5D)">Modulation Of Megakaryopoiesis And Platelet Production During Inflammation.</a> Thrombosis Research. 179, 114-120 (2019).</li>
<li>Kosaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vivo+Platelet+Production+From+Mature+Megakaryocytes%3A+Does+Platelet+Release+Occur+Via+Proplatelets%5BTitle%5D)+AND+%22International+Journal+Of+Hematology%22%5BJournal%5D)">In Vivo Platelet Production From Mature Megakaryocytes: Does Platelet Release Occur Via Proplatelets.</a> International Journal Of Hematology. 81 (3), 208-219 (2005).</li>
<li>Lefrancais, E., Looney, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet+Biogenesis+In+The+Lung+Circulation%5BTitle%5D)+AND+%22Physiology+%28Bethesda%29%22%5BJournal%5D)">Platelet Biogenesis In The Lung Circulation.</a> Physiology (Bethesda). 34 (6), 392-401 (2019).</li>
<li>Nieswandt, B., Stritt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Megakaryocyte+Rupture+For+Acute+Platelet+Needs%5BTitle%5D)+AND+%22Journal+Of+Cell+Biology%22%5BJournal%5D)">Megakaryocyte Rupture For Acute Platelet Needs.</a> Journal Of Cell Biology. 209 (3), 327-328 (2015).</li>
<li>Nishimura, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IL-1alpha+Induces+Thrombopoiesis+Through+Megakaryocyte+Rupture+In+Response+To+Acute+Platelet+Needs%5BTitle%5D)+AND+%22Journal+Of+Cell+Biology%22%5BJournal%5D)">IL-1alpha Induces Thrombopoiesis Through Megakaryocyte Rupture In Response To Acute Platelet Needs.</a> Journal Of Cell Biology. 209 (3), 453-466 (2015).</li>
<li>Sanjuan-Pla, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet-Biased+Stem+Cells+Reside+At+The+Apex+Of+The+Haematopoietic+Stem-Cell+Hierarchy%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Platelet-Biased Stem Cells Reside At The Apex Of The Haematopoietic Stem-Cell Hierarchy.</a> Nature. 502 (7470), 232-236 (2013).</li>
<li>Notta, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+Routes+Of+Lineage+Development+Reshape+The+Human+Blood+Hierarchy+Across+Ontogeny%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Distinct Routes Of Lineage Development Reshape The Human Blood Hierarchy Across Ontogeny.</a> Science. 351 (6269), 2116(2016).</li>
<li>Yamamoto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonal+Analysis+Unveils+Self-Renewing+Lineage-Restricted+Progenitors+Generated+Directly+From+Hematopoietic+Stem+Cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Clonal Analysis Unveils Self-Renewing Lineage-Restricted Progenitors Generated Directly From Hematopoietic Stem Cells.</a> Cell. 154 (5), 1112-1126 (2013).</li>
<li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decoding+Human+Megakaryocyte+Development%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Decoding Human Megakaryocyte Development.</a> Cell Stem Cell. , (2020).</li>
<li>Meinders, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repercussion+Of+Megakaryocyte-Specific+Gata1+Loss+On+Megakaryopoiesis+And+The+Hematopoietic+Precursor+Compartment%5BTitle%5D)+AND+%22Plos+One%22%5BJournal%5D)">Repercussion Of Megakaryocyte-Specific Gata1 Loss On Megakaryopoiesis And The Hematopoietic Precursor Compartment.</a> Plos One. 11 (5), 0154342(2016).</li>
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<li>Salunkhe, V. P., Gutiérrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+Of+Megakaryocytes+From+Human+Peripheral+Blood+Mononuclear+Cells%5BTitle%5D)+AND+%22Bio-Protocol%22%5BJournal%5D)">Culture Of Megakaryocytes From Human Peripheral Blood Mononuclear Cells.</a> Bio-Protocol. 5 (21), 1639(2015).</li>
<li>Choudry, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+Characterization+Of+Human+Megakaryocyte+Polyploidization+And+Lineage+Commitment%5BTitle%5D)+AND+%22Journal+Of+Thrombosis+And+Haemostasis%22%5BJournal%5D)">Transcriptional Characterization Of Human Megakaryocyte Polyploidization And Lineage Commitment.</a> Journal Of Thrombosis And Haemostasis. , 15271(2021).</li>
<li>Heazlewood, S. Y., Williams, B., Storan, M. J., Nilsson, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Prospective+Isolation+Of+Viable%2C+High+Ploidy+Megakaryocytes+From+Adult+Murine+Bone+Marrow+By+Fluorescence+Activated+Cell+Sorting%5BTitle%5D)+AND+%22Methods+In+Molecular+Biology%22%5BJournal%5D)">The Prospective Isolation Of Viable, High Ploidy Megakaryocytes From Adult Murine Bone Marrow By Fluorescence Activated Cell Sorting.</a> Methods In Molecular Biology. 1035, 121-133 (2013).</li>
<li>Tomer, A., Harker, L. A., Burstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+Of+Human+Megakaryocytes+By+Fluorescence-Activated+Cell+Sorting%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Purification Of Human Megakaryocytes By Fluorescence-Activated Cell Sorting.</a> Blood. 70 (6), 1735-1742 (1987).</li>
<li>Martinez-Botia, P., Acebes-Huerta, A., Seghatchian, J., Gutierrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+The+Quest+For+In+Vitro+Platelet+Production+By+Re-Tailoring+The+Concepts+Of+Megakaryocyte+Differentiation%5BTitle%5D)+AND+%22Medicina%22%5BJournal%5D)">On The Quest For In Vitro Platelet Production By Re-Tailoring The Concepts Of Megakaryocyte Differentiation.</a> Medicina. 56 (12), Kaunas. (2020).</li>
<li>Martinez-Botia, P., Acebes-Huerta, A., Seghatchian, J., Gutierrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vitro+Platelet+Production+For+Transfusion+Purposes%3A+Where+Are+We+Now%5BTitle%5D)+AND+%22Transfusion+And+Apheresis+Science%22%5BJournal%5D)">In Vitro Platelet Production For Transfusion Purposes: Where Are We Now.</a> Transfusion And Apheresis Science. 59 (4), 102864(2020).</li>
<li>Butov, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vitro+Megakaryocyte+Culture+From+Human+Bone+Marrow+Aspirates+As+A+Research+And+Diagnostic+Tool%5BTitle%5D)+AND+%22Platelets%22%5BJournal%5D)">In Vitro Megakaryocyte Culture From Human Bone Marrow Aspirates As A Research And Diagnostic Tool.</a> Platelets. , 1-8 (2020).</li>
<li>Di Buduo, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Gold+Standard+Protocol+For+Human+Megakaryocyte+Culture+Based+On+The+Analysis+Of+1%2C500+Umbilical+Cord+Blood+Samples%5BTitle%5D)+AND+%22Thrombosis+And+Haemostasis%22%5BJournal%5D)">A Gold Standard Protocol For Human Megakaryocyte Culture Based On The Analysis Of 1,500 Umbilical Cord Blood Samples.</a> Thrombosis And Haemostasis. , (2020).</li>
</ol>

Audiovisual material production was supported by BD Biosciences.

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 surface markers (Tables 1 and 2) in the same flow cytometry antibody panel for the study of megakaryopoiesis in a more detailed way. Our novel approach could serve as a powerful tool to aid diagnosis and/or prognosis in hematological disorders. We consider that the presented panels and combinations are not definitive. We have observed a very dynamic behavior of surface markers in human primary sources (mainly in BM samples), which is also dependent on the subjacent pathology. More studies are required, and hopefully, the scientific community will start applying this approach as to feedback with observations from studies focusing on a single pathology and how megakaryopoiesis staging may aid at diagnosis or prognosis. The implementation of combined HSC and MK characterization in primary tissues, considering the diverse routes for MK commitment as commented in the introduction section, is of relevance and worth of further developments. The fact that we are also able to detect MKs in PBMCs is quite an observation. We also hypothesize that the analysis of MKs in this primary source will aid in disease diagnosis and prognosis, as a very relevant alternative to BM aspirates, since extraction of a blood sample is not as invasive. That aspect also requires development.
Despite the technical limitations associated to the biological characteristics of MKs (large size, osmotic fragility, stickiness) we were able to sort different maturation stages in several human MK sources, specifically in BM, PBMCs and PBMCs-derived cell cultures. Future studies will be conducted in order to analyze the peculiarities of megakaryopoiesis in other human sources such as cord blood and in different physio-pathological situations. In addition, the differentiation potential of each gated and sorted cell fraction should be evaluated in colony-forming unit (CFU) assays of MK progenitors (CFU-MK) as to objectively assess the differentiation potential of each population in the megakaryopoiesis journey. While surpassing the technical limitations that were once thought as to impede the sort of mature megakaryocytes, such a necessity still demands more perfection. On one hand, single-cell Omics would allow comprehensive studies when applying the immunophenotyping panels, but if physical sort is required, despite the purity of the sorted populations is above 85% (based on surface marker expression), other variables need to be taken into account, which are still difficult to solve. As mentioned, MK cultures are not synchronous and are very heterogeneous. MKs themselves are promiscuous in surface marker expression, sharing markers with other myeloid and non-myeloid hematopoietic and non-hematopoietic cells. Furthermore, they are very difficult to cluster with the morphometric variables (Forward and Side Scatter) as they distribute widely. Further studies are required to refine this application, but with collaborative efforts, we are closer to narrow down megakaryopoiesis.

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><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&amp;uuml;nwald Giemsa</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; 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>&amp;nbsp;R&amp;amp;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&amp;trade;</td><td>PHC2115</td><td>or similar</td></tr><tr><td>Recombinant human thrombopoietin (TPO)</td><td>Thermo Fisher Scientific, Gibco&amp;trade;</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, &amp;kappa;/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>Note: we do not specify general reagents/chemicals (PBS, EDTA, etc) or disposables (tubes, etc), or reagents specified in previous published and standard protocols&amp;nbsp; - unless otherwise specified.</td><td></td><td></td><td></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.

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 ...

immunophenotyping-cell-sorting-human-mks-from-human-primary-sources

Nanyang Technological University

Research

JoVE Journal

Biology

6.5K Views.  Instituto de Investigación Sanitaria del Principado de Asturias (ISPA). Hallo.Ik ben Andrea Acebes Huerta.Postdoctoraal Onderzoeker bij het Platelet Research Lab onderdeel van het Health Research Institute of the Principality of Asturias, ISPA. Die is gevestigd in Oviedo, Spanje.Hello.I ben Laura Guti

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

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Genetics

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Developmental Biology

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Immunology and Infection

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

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 constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

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

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

Isolation of Mouse Megakaryocyte Progenitors

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through ...

Fluorescence-Activated Single-Cell Sorting of Human Mesenchymal Stem Cells

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and are known for their immunomodulatory and anti-inflammatory properties. The use of these stem cells is a boon to the field of regenerative biology, but at the same time, a bane to regenerative medicine and therapeutics owing to the multiple cellular ambiguities associated with them. These ambiguities may arise from the diversity in the source of these stem cells and from their in vitro growth conditions, both of which reflect upon their functional heterogeneity.
This warrants methodologies to provide purified, homogeneous populations of MSCs&#160;for therapeutic applications. Advances in the field of flow cytometry have enabled the detection of single-cell populations using a multiparametric approach. This protocol outlines a way to identify and purify&#160;stem cells from&#160;human exfoliated deciduous teeth (SHEDs) through fluorescence-assisted single-cell sorting. Simultaneous expression of surface markers, namely, CD90-fluorescein isothiocyanate (FITC), CD73-peridinin-chlorophyll-protein (PerCP-Cy5.5), CD105-allophycocyanin (APC), and CD44-V450, identified the &#34;bright,&#34; positive-expressors of MSCs&#160;using multiparametric&#160;flow cytometry. However, a significant drop was observed in percentages of quadruple expressors of these positive markers from passage 7 onwards to the later passages.
The immunophenotyped subpopulations were sorted using the single-cell sort mode where only two positive and one negative marker constituted the inclusion criteria. This methodology ensured the cell viability of the sorted populations and maintained cell proliferation post sorting. The downstream application for such sorting can be used to evaluate lineage-specific differentiation for the gated subpopulations. This approach can be applied to other single-cell systems to improve isolation conditions and for acquiring multiple cell surface marker information.

Author Spotlight: Importance of Single Cell Sorting in Isolating Purified Populations of Mesenchymal Stem Cells 

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and ...

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

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