Agradecemos a Marcos Pérez Basterrechea, Lorena Rodríguez Lorenzo e Begoña García Méndez (HUCA) e Paloma Cerezo, Almudena Payero e María de la Poveda-Colomo (HCSC) pelo apoio técnico. Este trabalho foi parcialmente apoiado por Bolsas Médicas (Roche SP200221001) para A.B., uma bolsa da RYC (RYC-2013-12587; Ministerio de Economía y Competitividad, Espanha) e uma subvenção I+D 2017 (SAF2017-85489-P; Ministerio de Ciencia, Innovación y Universidades, Espanha e Fondos FEDER) para L.G., um Subsídio Severo Ochoa (PA-20-PF-BP19-014; Consejería de Ciencia, Innovación y Universidades del Principado de Astúrias, Espanha) para P.M.-B. e uma bolsa de pós-doutorado intramuros 2018 (Fundación para la Investigación y la Innovación Biosanitaria de Astúrias - FINBA, Oviedo, Espanha) para A.A.-H. Agradecemos a Reinier van der Linden por compartilhar seu conhecimento (e tempo), especialmente seus sábios conselhos sobre a mistura de painel de anticorpos com várias cores e a preparação do controle de contas de cor única.

<ol>
	<li>Italiano, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+Mechanisms+That+Control+Platelet+Production.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Incredible+Journey:+From+Megakaryocyte+Development+To+Platelet+Formation.">The Incredible Journey: From Megakaryocyte Development To Platelet Formation.</a> Journal Of Cell Biology. 201 (6), 785-796 (2013).</li><li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+Of+The+Demarcation+Membrane+System+(DMS)+In+Megakaryocytes.">Biogenesis Of The Demarcation Membrane System (DMS) In Megakaryocytes.</a> Blood. 123 (6), 921-930 (2014).</li><li>Couldwell, G., Machlus, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+Of+Megakaryopoiesis+And+Platelet+Production+During+Inflammation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Platelet+Production+From+Mature+Megakaryocytes:+Does+Platelet+Release+Occur+Via+Proplatelets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+Biogenesis+In+The+Lung+Circulation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megakaryocyte+Rupture+For+Acute+Platelet+Needs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1alpha+Induces+Thrombopoiesis+Through+Megakaryocyte+Rupture+In+Response+To+Acute+Platelet+Needs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-Biased+Stem+Cells+Reside+At+The+Apex+Of+The+Haematopoietic+Stem-Cell+Hierarchy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Routes+Of+Lineage+Development+Reshape+The+Human+Blood+Hierarchy+Across+Ontogeny.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+Analysis+Unveils+Self-Renewing+Lineage-Restricted+Progenitors+Generated+Directly+From+Hematopoietic+Stem+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+Human+Megakaryocyte+Development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repercussion+Of+Megakaryocyte-Specific+Gata1+Loss+On+Megakaryopoiesis+And+The+Hematopoietic+Precursor+Compartment.">Repercussion Of Megakaryocyte-Specific Gata1 Loss On Megakaryopoiesis And The Hematopoietic Precursor Compartment.</a> Plos One. 11 (5), 0154342 (2016).</li><li>Meinders, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sp1/Sp3+Transcription+Factors+Regulate+Hallmarks+Of+Megakaryocyte+Maturation+And+Platelet+Formation+And+Function.">Sp1/Sp3 Transcription Factors Regulate Hallmarks Of Megakaryocyte Maturation And Platelet Formation And Function.</a> Blood. 125 (12), 1957-1967 (2015).</li><li>Salunkhe, V. P., Guti&#233;rrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+Of+Megakaryocytes+From+Human+Peripheral+Blood+Mononuclear+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Characterization+Of+Human+Megakaryocyte+Polyploidization+And+Lineage+Commitment.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Prospective+Isolation+Of+Viable,+High+Ploidy+Megakaryocytes+From+Adult+Murine+Bone+Marrow+By+Fluorescence+Activated+Cell+Sorting.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+Of+Human+Megakaryocytes+By+Fluorescence-Activated+Cell+Sorting.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+The+Quest+For+In+Vitro+Platelet+Production+By+Re-Tailoring+The+Concepts+Of+Megakaryocyte+Differentiation.">On The Quest For In Vitro Platelet Production By Re-Tailoring The Concepts Of Megakaryocyte Differentiation.</a> Medicina. 56 (12), (2020).</li><li>Martinez-Botia, P., Acebes-Huerta, A., Seghatchian, J., Gutierrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Platelet+Production+For+Transfusion+Purposes:+Where+Are+We+Now.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Megakaryocyte+Culture+From+Human+Bone+Marrow+Aspirates+As+A+Research+And+Diagnostic+Tool.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Gold+Standard+Protocol+For+Human+Megakaryocyte+Culture+Based+On+The+Analysis+Of+1,500+Umbilical+Cord+Blood+Samples.">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>

A produção de material audiovisual foi apoiada pela BD Biosciences.

A maior parte da pesquisa com foco no estudo da megacariopoiese por citometria de fluxo é até hoje limitada à identificação de subconjuntos MK usando apenas marcadores de superfície específicos de linhagem (ou seja,CD42A/CD42B, CD41/CD61), enquanto estágios anteriores de diferenciação de MK foram mal examinados. No presente artigo mostramos uma estratégia de imunofenofoteping para abordar uma caracterização abrangente da citometria de fluxo da megacariopoiese humana. No geral, gostaríamos de destac...

Os megacariócitos (MKs) desenvolvem-se a partir de células-tronco hematopoiéticas (HSCs) seguindo um processo complexo chamado megacariiopoiese, que é orquestrado principalmente pelo hormônio trombopoietina (TPO). A visão clássica da megacaripoiese descreve a jornada celular dos HSCs através de uma sucessão de estágios hierárquicos de progenitores comprometidos e células precursoras, levando finalmente a um MK maduro. Durante a maturação, os MKs experimentam múltiplas rodadas de endomitose, desenvolvem um intrincado sistema de membrana intracelular (DMS), que fornece superfície de membrana suficiente para a produção de plaquetas, e produzem e embalam eficientemente a infinidade de fatores que estão contidos nos diferentes grânulos herdados pelas plaquetas maduras1,2,3. Como resultado, MKs maduros são células grandes (40-60 μm) caracterizadas por um núcleo altamente poliploide (atingindo até mesmo >64N). Estudos recentes sugerem rotas alternativas pelas quais os HSCs se diferenciam em MKs contornando os pontos de verificação tradicionais de compromisso de linhagem em resposta a certas condições fisio-patológicas4,5,6,7,8,9,10,11. Esses achados destacam que a diferenciação hematopoiética em relação ao MK maduro é um processo contínuo e adaptativo que responde às necessidades biológicas.
Com o crescente conhecimento sobre a biologia celular e os aspectos moleculares caracterizando a megacariopoiese12, a maioria das pesquisas dedicadas ao estudo do processo por citometria de fluxo estão limitadas à identificação de MKs maduros usando marcadores de superfície específicos de linhagem (ou seja, CD42A/B, CD41/CD61), enquanto os estágios anteriores de diferenciação de MK permanecem inexplorados. Documentamos anteriormente uma estratégia para encenar megacaripoiesis em medula óssea de camundongos e culturas MK derivadas da medula óssea13,14, que adaptamos e aplicamos aos humanos15. No presente artigo mostramos uma estratégia de imunofenofoteca que permite a caracterização da megacariiopoiese, dos HSCs aos MKs maduros, em fontes primárias humanas (medula óssea -BM e sangue periférico -PB-) ou culturas in vitro utilizando um painel integrando marcadores de superfície específicos e não específicos MK (CD61, CD42B, CD49B, CD31, KIT e CD71, entre outros). Apesar de seu grande tamanho e fragilidade, os MKs podem ser imunofenótipados usando os marcadores de superfície celular acima mencionados e enriquecidos pela classificação celular ativada pela fluorescência em condições específicas de pressão e diâmetro do bocal para minimizar a ruptura celular e/ou danos. Essa técnica facilita abordagens multi-Omics, com o objetivo de compreender melhor a complexidade da megacariopoiese e da produção de plaquetas na saúde humana e na doença. Vale ressaltar que será uma ferramenta útil para auxiliar o diagnóstico e o prognóstico em um contexto clínico de crescente demanda.
Neste manuscrito documentamos uma estratégia para encenar megacaripoiesis humanas com um painel integrando marcadores de superfície específicos e não específicos de MK de fontes primárias ou gerados in vitro. Além disso, fornecemos um protocolo para classificar, com um classificador celular ativado por fluorescência, as frações preferidas e MKs maduros(Figura 1). Este passo não é popular, pois é tecnicamente difícil devido ao grande tamanho e fragilidade dos MKs. No entanto, tem sido empregado tanto em amostras de camundongos quanto de medula óssea humana anteriormente, e devido ao avanço tecnológico, com melhor resultado a cada vez16,17,18. Fontes primárias humanas onde mks ou precursores mk podem ser estudados incluem medula óssea, sangue do cordão umbilical e sangue periférico, entre outros. O processamento adequado da amostra para isolar a fração celular relevante para análise em cada amostra é de importância. Os procedimentos padrão são incorporados, com algumas considerações a serem consideradas ao mirar no estudo da megacaripoiese.

<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

A diferenciação de megacariócito (MK) abrange uma série de ciclos endomitóticos que resultam em uma célula altamente poliploide (atingindo até mesmo >64N) e extremamente grande (40-60 μm). Ao contrário do rápido crescimento do conhecimento em megacaripoiesis na biologia celular e no nível molecular, a caracterização da megacariopoiese por citometria de fluxo limita-se à identificação de MKs maduros usando marcadores de superfície específicos da linhagem, enquanto os estágios anteriores de diferenciação de MK permanecem inexplorados. Aqui, apresentamos uma estratégia de imunofenofoteping que permite a identificação de estágios sucessivos de diferenciação de MK, com status ploidy crescente, em fontes primárias humanas ou culturas in vitro com um painel integrando marcadores de superfície específicos e não específicos de MK. Apesar de seu tamanho e fragilidade, os MKs podem ser imunofenótipados usando o painel acima mencionado e enriquecidos pela triagem celular ativada pela fluorescência em condições específicas de pressão e diâmetro do bocal. Essa abordagem facilita estudos multi-Omics, com o objetivo de entender melhor a complexidade da megacariopoiese e da produção de plaquetas em humanos. Uma melhor caracterização da megacaritopoiese pode representar fundamental no diagnóstico ou prognóstico de patologias e malignidade relacionadas à linhagem.

Medula Óssea e Ploidy Na Figura 4, mostramos uma análise representativa de imunofenofoteping de megacaripoiese em amostras de BM (aspiração) de pacientes. Ao traçar a fração celular contra CD71 e CD31, temos fechado seis populações principais: CD31- CD71- (vermelho), CD31- CD71+ (azul), CD31+ CD71- (laranja), CD31+ CD71mid (verde claro), 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

Aqui, apresentamos uma estratégia de imunofenofoteping para a caracterização da diferenciação de megacaitos, e mostramos como essa estratégia permite a classificação de megacaiócitos em diferentes estágios com um classificador celular ativado pela fluorescência. A metodologia pode ser aplicada aos tecidos primários humanos, mas também aos megacaitos gerados na cultura in vitro.

Imunofenoftipagem e Triagem celular de MKs humanos de fontes primárias humanas ou in vitro diferenciados de progenitores hematopoiéticos

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

Olá.Sou Andrea Acebes Huerta.Pesquisadora de pós-doutorado no Laboratório de Pesquisa plaqueteira do Instituto de Pesquisa em Saúde do Principado das Astúrias, ISPA. Que está localizada em Oviedo, Espanha.Olá.Eu sou Laura Guti

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

Research

JoVE Journal

Biology

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

6.3K visualizações.  Instituto de Investigación Sanitaria del Principado de Asturias (ISPA). Olá.Sou Andrea Acebes Huerta.Pesquisadora de pós-doutorado no Laboratório de Pesquisa plaqueteira do Instituto de Pesquisa em Saúde do Principado das Astúrias, ISPA. Que está localizada em Oviedo, Espanha.Olá.Eu sou Laura Guti

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

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 MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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

Biologia

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 function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Primárias células epiteliais brônquicas humanas Grown a partir de explantes

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 transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivação eficiente dos recursos humanos Precursores cardíaca e Cardiomyocytes pluripotentes a partir de células estaminais embrionárias humanas com a indução de moléculas pequenas

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 ...

Na&iuml;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 was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

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

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento de culturas primárias de fibroblastos humanos

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 functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Isolamento de primárias Miofibroblastos de Mouse e tecido do cólon humano

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 include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente Geração induzida pelo homem células-tronco pluripotentes a partir de células somáticas humanas com Sendai-vírus

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

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

In Vitro Pâncreas Organogenesis de dispersos rato embrionárias Progenitores

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

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolamento e Cultura de Queratinócitos Orais Primários do Paladar do Rato Adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Classificação de célula única ativada por fluorescência de células-tronco mesenquimais humanas

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.

Autor em destaque: Importância da classificação de células únicas no isolamento de populações purificadas de células-tronco mesenquimais 

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.

Triagem unicelular de células-tronco mesenquimais imunofenotipadas de dentes decíduos esfoliados humanos

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