Os autores reconhecem o apoio da saúde australiano e Medical Research Council (projeto concessão 1012409 ligado ao BHC).

Este protocolo foi aprovado pelo sul oriental Sydney humano pesquisa Comitê de ética e ratificado pela Comissão de ética de pesquisa humana a Universidade de New South Wales'. Sangue de cordão umbilical obtido de doadores saudáveis foi fornecido por o Sydney banco de sangue (Sydney, NSW, Austrália). Volumes de cerca de 100 mL foram utilizados para este procedimento.
Nota: Trabalho em uma segurança biológica classe II gabinete utilizando técnica asséptica. Descontaminar o exterior do saco de sangue de cordão com etanol a 70%. Use instrumentos esterilizados (tesouras, pinças) para este procedimento.
1. cabo de isolamento de CD34 e preparação de glóbulos+ células
<ol>
	<li>Prepare o tampão estéril de separação (SB) com solução salina tamponada fosfato (PBS), em pH 7,2 e contendo 0,5% bovina albumina de soro e 2 mM EDTA.</li>
	<li>Dispense 10 mL de SB em um tubo cónico de 50 mL (um tubo por 10 mL de sangue é necessário). Usando uma agulha montada em uma seringa de 10 mL G18, retirar 10 mL de sangue do saco e distribuir para os tubos de 50 mL contendo 10 mL de SB.</li>
	<li>Adicione 15 mL de mídia de separação de linfócitos (ver Tabela de materiais) para o fundo do tubo contendo o sangue diluído, criando duas camadas. Observação: Consomem mídia lentamente na parte inferior do tubo para evitar misturar as diferentes camadas.</li>
	<li>Centrifugar tubos em 1.200 × g por 30 min à temperatura ambiente (RT) sem interrupção e sem aceleração.</li>
	<li>Transferi a camada perto do centro do tubo contendo células mononucleares (aproximadamente 5-10 mL de cada tubo) em um novo tubo de 50 mL com uma pipeta Pasteur. Adicione SB para cada tubo para um volume total de 50 mL.</li>
	<li>Centrifugar a 400 × g por 10 min a RT. descartar o sobrenadante.</li>
	<li>Resuspenda pelotas célula cuidadosamente com uma pipeta Pasteur em 5-10 mL de SB Combine as células suspensas e trazer volume para 50 mL com SB.</li>
	<li>Contagem de células usando Trypan azul coloração e um hemocytometer ou automatizada de células contador (ver Tabela de materiais). Para determinar a porcentagem de CD34+ células na amostra, Ressuspender 2,5 × 105 em 100 µ l de SB e mancha, adicionando 10 µ l de anticorpo anti-CD34-PE por 15-30 min a 4 ° C. Análise por citometria de fluxo (figura 1A).</li>
	<li>Tubos de centrífuga a 400 × g por 10 min a 4 ° C. Descarte o sobrenadante. Nota: se não for necessário imediatamente, células mononucleares podem ser congeladas nesta fase.</li>
	<li>Ressuspender as células em 300 µ l de SB por 108 células. Adicione 100 µ l de IgG humana FcR (bloqueio de reagente, consulte Tabela de materiais) e 100 µ l de CD34 grânulos magnéticos por 108 células. Misture delicadamente e incubar a 4 ° C por 30-40 min. Nota: Uma suspensão de célula única é necessária (se necessário, passar as células embora um filtro 30 µm antes de adicionar os reagentes). Use os volumes de reagente descritos aqui por 10 células de8 ou menos. Para mais de 108 células, aumenta os reagentes (SB, bloqueando a solução e grânulos magnéticos CD34) conformemente.</li>
	<li>Prepare a coluna de separação é durante o período de incubação, colocando a coluna de LS em um suporte magnético e o efluente de lavagem com 3 mL de SB Discard. Nota: Colunas de separação de LS podem ser carregadas com até 2 × 108 células total. Colunas maiores e menores estão disponíveis.</li>
	<li>Adicionar 5-10 mL de SB para a mistura de células e centrifugar 400 × g por 10 min. descartar o sobrenadante.</li>
	<li>Ressuspender as células em 1,5 mL de SB e carregar a suspensão de eritrócitos (1,5 mL) para a coluna de LS. Colete o fluxo que contém as células sem rótulo em um tubo de coleta de 15 mL (fração negativa #1). Deixe o líquido escorrer e lavar a coluna com 1,5 mL de SB.</li>
	<li>Carregar o fluxo coletado através de (3 mL) volta para a coluna e coletar o fluxo através do tubo de coleta 15 mL mesmo (fração negativa #1). Lave a coluna de LS 3 vezes com 3 mL de SB. Nota: Se necessário, as células na fração negativa (12 mL) podem ser manchadas com anticorpo anti-CD34 para apurar a captura de CD34+ células pela coluna LS.</li>
	<li>Remover coluna a partir do separador magnético e irrigue células lentamente com um êmbolo da seringa para um novo tubo de 15 mL com 2 mL de SB.</li>
	<li>Coloque a coluna volta no separador magnético e carga a 2 mL de células volta para a coluna. Coletar o fluxo através de e lavar com 2 mL SB. Esta é a fração negativa #2 (4 mL).</li>
	<li>Remover coluna LS de separador magnético e adicionar 2 mL de SB Flush células firmemente e firmemente com um êmbolo da seringa para coletar o CD34+ fração de célula. Conte as células com coloração azul trypan e um hemocytometer ou contador automatizado de células.</li>
	<li>Tubo de centrífuga com a fração positiva a 400 × g por 15 min a 4 ° C. Desprezar o sobrenadante. Ressuspender as células em media serum-free para CD34+ células (SFM, consulte Tabela de materiais). Nota: se não for necessário, imediatamente, as células podem ser congeladas em nitrogênio líquido nesta fase.</li>
</ol>
2. seleção pureza de CD34 isoladas células+
<ol>
	<li>Mancha de 2 x 104 células da fracção positiva com 10 µ l anti-CD34-PE anticorpo e anticorpo anti-CD45-PerCP de 20 µ l por 15 min a 4 ° C (use um separado tubo para controles do isotipo) (figura 1B).</li>
	<li>Adicione 1 mL de SB para lavar. Centrifugar a 400 × g por 10 min. Ressuspender pelota em 200 µ l de SB.</li>
	<li>Realizar análise de fluxo cytometric e portão a população de células vivas para excluir os detritos (figura 1B). Definir o portão para PE PerCP positivo as populações e usando os controles de isotipo PE e PerCP (figura 1B) e determinar a porcentagem de CD34+/CD45+ células.</li>
</ol>
3. megacariócito diferenciação
<ol>
	<li>Semente de 5 × 105 células/mL CD34+ em 2 mL de SFM suplementado com 50 ng/mL Trombopoietina humana recombinante (rhTPO) por bem em uma placa de 12 células. Incube as células a 37 ° C, 5% de CO2 em um ambiente umidificado. Se as células são confluentes, antes que eles são necessários para a análise, colher as células e dividir em vários poços com mídia fresca e rhTPO. Nota: Dois ou três poços devem ser preparados especificamente para monitorar a diferenciação em pontos de tempo diferente (por exemplo, dias 7, 9 e 10).</li>
	<li>Colher células dos poços reservou para monitorar diferenciação sem perturbar as células em outros poços.</li>
	<li>Mancha de células com anticorpo anti-GPIIb/CD41-FITC de 20 µ l e 10 µ l anti-GPIX/CD42a-Alexa Fluor 647 anticorpo um volume final de 100 µ l. configurar um controle tubo usando anticorpos respectivos do isotipo controle. Incubar durante 15-30 min a 4 ° C.</li>
	<li>Adicione 1 mL de SB para lavar.</li></ol>Centrifugar a 400 × g por 10 min. descartar o sobrenadante e ressuspender o precipitado em 200-300 µ l de SB Analyze por citometria de fluxo.
	<li>Para cada fluoróforo, analise os isotipo controles para definir o canal para FITC e Alexa farinha 647 populações positivas. Analisar as amostras de células coradas para determinar a porcentagem de CD41+/CD42a+ duplo células positivas, que representam MK maduro (Figura 1).</li>
	<li>Para visualização microscópica de célula marcadores de superfície mancham células conforme descrito no ponto 3.3.<ol>
		<li>Adicione 1 mL de SB para lavar. Centrifugar a 400 × g por 10 min. resuspenda o pellet em 100 µ l de SB e girar numa lâmina de vidro a 1.000 x g, durante 5 min. Fix células no slide mergulhando em metanol por 30 s. ar seco, adicionar 20 µ l de mídia contendo DAPI de montagem (ver Tabela de materiais) , cobrir com uma lamela e Visualizar usando um microscópio fluorescente (Figura 2A).</li>
	</ol>
	</li>
	<li>Para visualização dos antígenos intracelulares, Ressuspender as células em PBS e corrigir com paraformaldeído (concentração final de 1%) durante 15 minutos à temperatura ambiente. Para permeabilize as células, adicionar o triton-X100 (0,1%) e incube por 15 min. células de lavagem com 2 mL PBS/0.1% triton-X100. Resuspenda em 100 µ l de PBS/0.1% triton-X100, adicionar anti vWf e anti CD62p anticorpos (diluição de 1: 200) e incube por 30 min à temperatura ambiente.<ol>
		<li>Lavar com 2 mL PBS/0.1% triton-X100 e centrifugar a 400 × g por 10 min, resuspenda em 100 µ l de buffer mesmo e adicionar anti-mouse IgG-Alexa 594 e anti-coelho IgG-Alexa 488 (1: 100). Incubar durante 30 min à temperatura ambiente, lave com 2 mL PBS/0.1% triton-X100, resuspenda em 100 µ l de buffer mesmo e adicionar 20 µ l de anti CD42b-APC. Em seguida girar as células em lâminas de vidro, conforme descrito na etapa 3.6.1 e preparar amostras para visualização microscópica, conforme descrito na etapa 3.6.1.</li>
	</ol>
	</li>
	<li>Para determinação da ploidia, colha células no dias 12 ou 13 de diferenciação. Adicione 1 mL de SB para lavar. Centrifugar a 400 × g por 10 min e mancha com anticorpo de anti-GPIIb/CD41-FITC 20 µ l em um volume final de 100 µ l. Incubar a 4 ° C por 30 min.<ol>
		<li>Lave uma vez com 1 mL de SB e resuspenda o pellet em 300 µ l de citrato hipotônica tampão (citrato de sódio 1,25 mM, 2,5 mM de cloreto de sódio, glicose de 3,5 mM) contendo 20 µ g/ml propidium iodeto e 0,05% Triton X 100. Incube por 15 min a 4 ° C, protegido da luz.</li>
		<li>Adicionar RNase a uma concentração final de 20 µ g/mL e incube por 30 min a 4 ° C, protegido da luz. Determine a intensidade de iodeto de propidium por citometria de fluxo através da recolha de 30.000 a 50.000 eventos da população CD41-FITC+ (Figura 2).</li>
	</ol>
	</li>

4. proplatelet contagem, Enumeração plaquetária e Ativação plaquetária
<ol>
	<li>Colheita de células (da etapa 3.1) cada dias 8 ou 9 da diferenciação e da semente no 1 × 104 células/poço em placas boas 48 em 200 µ l de SFM fresco suplementado com 50 ng/mL rhTPO. Cultura durante 5 dias a 37 ° C, 5% CO2. Nota: Para fins de quantificação, semente wells em triplicado. Esta baixa densidade é necessária para a visualização e contagem dos proplatelet-rolamento Proplatelets MK geralmente começam a aparecer depois de 2 dias de cultura. O pico é entre os dias 4 e 5.</li>
	<li>Conte o número de proplatelet-rolamento MK no poço inteiro em um microscópio invertido usando 10 X ou 20 X de objectivos. Nota: Um palco de microscópio aquecida (37 ° C) é preferível, desde que manter as células à temperatura ambiente por períodos prolongados provoca encolhimento das extensões de proplatelet. Proplatelets são observados como longas extensões do corpo MK. Cada MK pode ter várias saliências proplatelet. Como desenvolver proplatelets, o corpo do MK diminui de tamanho.</li>
	<li>Colheita de células e centrifugar a 400 x g durante 10 minutos à temperatura ambiente. Mancha de células com anticorpo anti-humano CD41-FITC 20 µ l como descrito nos passos 3.3 e 3.4. Calcular a porcentagem de MK proplatelet-rolamento (pbMK): pbMK (%) = [(Proplatelet-rolamento MKs/bem) / (Total CD41+ células / bem)] x 100</li>
	<li>Para contar as plaquetas lançadas no meio de cultura, delicadamente misture as células com uma pipeta Pasteur e coletar 100 µ l em dias 14 ou 15 da cultura.<ol>
		<li>Mancha com 20 µ l anti-humano CD41-FITC de anticorpos para 20-30 min a 4 ° C. Configure um tubo de controle usando o respectivo do isotipo controle anticorpo.</li>
		<li>Adicione 150 µ l de SB e 50 µ l de contagem de grânulos.</li>
		<li>Para análise de fluxo cytometric, defina o FSC e SSC para escala de log. Uso normais plaquetas sanguíneas humanas do plasma rico em plaquetas manchado com CD41-FITC, conforme descrito na seção 4.4.1 para definir o canal para plaquetas (Figura 3A).</li>
		<li>Analise as plaquetas manchadas com a contagem de grânulos por citometria de fluxo. Colete 1.000 eventos de contagem de contas usando o FSC contra SSC gráfico de dispersão (Figura 3A). Calcule o número de plaquetas com base em eventos positivos CD41-FITC (Figura 3B) usando a fórmula: Plaquetas por µ l = [(número de eventos positivos CD41-FITC)/1,000 beads)] x [(number of beads in 50 µL)/volume de amostra)]</li>
	</ol>
	</li>
	<li>Para analisar a ativação plaquetária, delicadamente misture as células com uma pipeta Pasteur e coletar 100 µ l em dias 14 ou 15 da cultura. Adicione 1 mL do Tyrode buffer (137 mM NaCl, 2,7 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 0,2 mM Na2HPO4, 12mm NaHCO3, 5,5 mM D-glicose, pH 6,5) e centrifugar a 200 x g por 5 min para células de Pelotas.<ol>
		<li>Recolha o sobrenadante e centrifugar a 800 x g durante 10 minutos para as plaquetas de tamanho de partículas de Pelotas.</li>
		<li>Descartar o sobrenadante e ressuspender em 100 µ l de tampão de Tyrode. Adicionar 20 µ l de anticorpo PAC1-FITC e difosfato de adenosina (ADP) a uma concentração final de 20 µM. Incubar à temperatura ambiente por 20 min. análise por citometria de fluxo para determinar a porcentagem de eventos positivos FITC. Uso de plaquetas humanas frescas trataram da mesma maneira como um controle positivo (Figura 3).</li>
	</ol>
	</li>
</ol>

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Os autores não têm nada para divulgar.

O protocolo descrito aqui é adequado para uma produção consistente de MK e plaquetas na cultura do sangue do cordão umbilical. Estas células podem ser usadas para estudar vários processos tais como o efeito de drogas ou atividades biológicas na MK proliferação, diferenciação, proplatelet de formação e produção de plaquetas.
Uma variedade de meios de cultura e combinações de citocinas foram apresentados na literatura. Adição de citocinas, como fator de células-tronco, ligant...

Isolamento de números adequados de megacariócitos humanos primários (MK) para uso em laboratório regular não é viável devido a sua baixa frequência na medula óssea, onde eles representam ~0.01% de células nucleadas1. Uma alternativa conveniente é a expansão ex vivo e diferenciação de tronco hematopoiético e células progenitoras na presença de fatores de crescimento específicos. Um número de citocinas, incluindo o fator de células-tronco (SCF; c-kit ligand) e interleucina (IL) -3 e IL-11 foram empregadas nos sistemas de cultura para produzir MKs. Thrombopoietin (TPO) é o fator mais eficaz de crescimento e diferenciação para culturas megakaryocytic e é eficaz, sozinho ou com outras citocinas, tais como SCF e IL-32. TPO pode agir sobre as populações de células-tronco para ocasionar a proliferação e a maturação dos MKs2.
E MK produzem plaquetas de protrusões citoplasmáticas, chamados proplatelets, in vivo, aproximadamente 1 x 1011 plaquetas formada diariamente para manter a contagem de plaquetas de 150-400 x 109plaquetas /L. produção em vitro é até 1000 -Dobre inferior a na vivo estima3, e isto tem dado origem a inúmeras condições de cultura usando CD34+ células progenitoras hematopoiéticas para melhorar MK e plaquetas produção em vitro. A fonte inicial de CD34+ células utilizadas para diferenciação de MK foi sangue periférico humano4. Outras fontes de célula incluem a medula óssea5,6, células-tronco embrionárias/induzida (ESC/iPSC) as células-tronco pluripotentes7e cordão umbilical sangue (UCB)8,9,10 . Medula óssea humana CD34+ 11 e mouse linhagem negativa da medula óssea células5 produtos MK e plaquetas in vitro; no entanto, a falta de disponibilidade de medula óssea humana limita a sua utilização como fonte de CD34+ células. Em contraste, ESC e iPSC representam uma fonte ilimitada de células para a produção de plaquetas em vitro . Produção de plaquetas a partir dessas células requer alimentador células como murino células OP9 e períodos mais longos de cultura. As plaquetas derivadas no alimentador-free condições parecem ser menos funcional12. iPSC-derivado de plaquetas são susceptíveis de ser de uso em ambientes clínicos desde que eles podem ser expandidos para grande escala. Este processo requer transdução mediada por Lentivirus de fatores de transcrição e de cultura de células a longo prazo13.
UCB é uma fonte acessível de CD34+ células que podem ser facilmente usadas em configurações de pesquisa. TPO sozinho pode promover a diferenciação da medula hemoderivados CD34+ células e isto dá origem a MKs altamente puro, maduro, sem a necessidade de suplementação de soro ou co-cultura com células do alimentador. Outras citocinas tais como SCF pode diminuir a diferenciação da UCB CD34+ células, enquanto o ligante Flt-3 e IL-11 promovem a produção de megacariócitos imaturo14. Este protocolo descreve a produção de culturas de MK altamente puras do sangue do cordão CD34+ células em condições de livre de soro.

<table><tbody><tr><td>&lt;strong&gt;Cell Culture Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Recombinant Human TPO</td><td>Miltenyi Biotec</td><td>130-094-013</td><td></td></tr><tr><td>StemSpan SFEM II</td><td>Stem Cell Technologies</td><td>9605</td><td>Serum-free media for CD34+ cells</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;CD34 Isolation Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD34 MicroBead kit ultrapure</td><td>Miltenyi Biotec</td><td>130-100-453</td><td>This kit includes the FcR human IgG blocking reagent and CD34 microbeads. These beads contain the anti-CD34 antibody clone QBEND/10. Use a different anti-CD34 clone for purity check (e.g. clone 8G12).</td></tr><tr><td>Lymphoprep</td><td>Alere Technologies</td><td>1114545</td><td>Lymphocyte separation media (density 1.077 g/mL)</td></tr><tr><td>Sterile separation buffer (SB)</td><td>Miltenyi Biotec</td><td>130-091-221</td><td>This buffer contains phosphate buffered saline (PBS), pH 7.2 containing 0.5% bovine serum albumin and 2 mM EDTA. It can be prepared using sterile, cell culture grade components. De-gas before use because air bubbles can block the column.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Flow Cytometry and Cell Staining Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PE Mouse anti-Human CD34</td><td>BD Biosciences</td><td>340669</td><td>Clone 8G12. This can be used for CD34 purity check. Final antibody concentration 1:10 dilution.</td></tr><tr><td>PerCP mouse anti-human CD45</td><td>BD Biosciences</td><td>347464</td><td>1:10 dilution</td></tr><tr><td>PerCP isotype control</td><td>BD Biosciences</td><td>349044</td><td>1:10 dilution</td></tr><tr><td>FITC Mouse anti-Human CD41a</td><td>BD Biosciences</td><td>340929</td><td>Final antibody concentration 1:5 dilution.</td></tr><tr><td>APC Mouse anti-Human CD42b</td><td>BD Biosciences</td><td>551061</td><td>This antibody can also be used to detect mature MK (the percentage of positive cells in usually lower than with anti CD42a). Final antibody concentration 1:10 dilution.</td></tr><tr><td>Alexa Fluor 647 Mouse anti-Human CD42a</td><td>AbD Serotec</td><td>MCA1227A647T</td><td>Currently distributed by Bio-Rad. Final antibody concentration 1:10 dilution.</td></tr><tr><td>Alexa Fluor 647 Mouse Negative Control</td><td>AbD Serotec</td><td>MCA928A647</td><td>Currently distributed by Bio-Rad. Isotype control antibody</td></tr><tr><td>Anti von Willebrand factor rabbit polyclonal</td><td>Abcam</td><td>AB6994</td><td>1:200 dilution</td></tr><tr><td>V450 mouse anti-humna CD41a</td><td>BD Biosciences</td><td>58425</td><td>1: 20 dilution</td></tr><tr><td>V450 isotype control</td><td>BD Biosciences</td><td>580373</td><td>1:20 dilution</td></tr><tr><td>PAC1-FITC antibody</td><td>BD Biosciences</td><td>340507</td><td>1:10 dilution</td></tr><tr><td>Anti CD62p mouse monoclonal</td><td>Abcam</td><td>AB6632</td><td>1:200 dilution</td></tr><tr><td>Alexa Fluor 488 goat anti rabbit IgG</td><td>Invitrogen</td><td>A11008</td><td>1:100 dilution</td></tr><tr><td>Alexa Fluor 594 goat anti mouse IgG</td><td>Invitrogen</td><td>A11020</td><td>1:100 dilution</td></tr><tr><td>Ig Isotype Control cocktail-C</td><td>BD Biosciences</td><td>558659</td><td>Isotype control antibody</td></tr><tr><td>Propidium iodide</td><td>Sigma Aldrich</td><td>P4864</td><td></td></tr><tr><td>CountBright Absolute Counting Beads</td><td>Molecular Probes, Invitrogen</td><td>C36950</td><td>Counting beads</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>Smaller and larger columns are also commercially available</td></tr><tr><td>MidiMACS Separator magnet</td><td>Miltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Falcon 5mL round bottom polypropylene FACS tubes, with Snap Cap, Sterile</td><td>In Vitro technologies</td><td>352063</td><td></td></tr><tr><td>Glass slides</td><td>Menzel-Glaser</td><td>J3800AMNZ</td><td></td></tr><tr><td>Mounting media with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td>Antifade mounting medium with DAPI</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Leica</td><td>DMIRB inverted microscope</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Zeiss</td><td>Vert.A1</td><td></td></tr><tr><td>Cell analyser</td><td>BD Biosciences</td><td>FACS Canto II</td><td></td></tr><tr><td>Cytospin centrifuge</td><td>ThermoScientific</td><td>Cytospin 4</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell analyser software</td><td>BD Biosciences</td><td>FACS Diva Software</td><td></td></tr><tr><td>Single cell analysis software</td><td>Tree Star</td><td>FlowJo</td><td></td></tr><tr><td>Fluorescent microscope software</td><td>Zeiss</td><td>Zen 2 blue edition</td><td></td></tr></tbody></table>

megakaryocyte differentiation and platelet formation from human cord blood-derived cd34+ cells

A produção de plaquetas ocorre principalmente na medula óssea em um processo conhecido como thrombopoiesis. Durante thrombopoiesis, células progenitoras hematopoiéticas diferenciam de precursores de plaquetas de formulário chamados megacariócitos, que diferenciam terminal para liberar as plaquetas de processos citoplasmáticos tempo denominados proplatelets. Megacariócitos são células raras confinadas à medula óssea e, portanto, são difíceis de colheita em número suficiente para uso em laboratório. Produção eficiente de megacariócitos humanas pode ser alcançada em vitro por cultivo CD34+ células sob condições apropriadas. O protocolo detalhado aqui descreve o isolamento de CD34+ células por célula magnética, classificação de amostras de sangue de cordão umbilical. São descritos os passos necessários para produzir megacariócitos altamente puros, maduros, em condições de livre de soro. Detalhes de análise fenotípica de diferenciação megacariócito e determinação da produção proplatelet de formação e plaquetas também são fornecidos. Efetores que influenciam a diferenciação do megacariócito e/ou formação proplatelet, tais como anticorpos anti-plaquetários ou mimetics trombopoietina, podem ser adicionados às células cultivadas para examinar a função biológica.

Este protocolo permite a preparação das culturas de MK altamente puras do cordão hemoderivados CD34+ células. A porcentagem de CD34+ células na medula sangue é, aproximadamente, 1,3%15 (figura 1A) e o número total de células mononucleares (passo 1.8) intervalos de 90-300 x 106 por unidade da UCB. A pureza do CD34 + / CD45 + células após intervalos de isolamento de 90 a 99% (<strong class="xfig...

Watch this Scientific Journal Video about Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells at JoVE.com

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

Uma população altamente pura de megacariócitos pode ser Obtida de cordão hemoderivados CD34+ células. Um método para CD34+ diferenciação de isolamento e megacariócito célula está descrita aqui.

Megacariócito diferenciação e formação de plaquetas de humanos cabo hemoderivados CD34 células+

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

megakaryocyte-differentiation-platelet-formation-from-human-cord

Research

JoVE Journal

Immunology and Infection

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

19.5K visualizações.  University of New South Wales. Uma população altamente pura de megacariócitos pode ser Obtida de cordão hemoderivados CD34+ células. Um método para CD34+ diferenciação de isolamento e megacariócito célula está descrita aqui.

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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Geração de Células T reguladoras induzidas a partir de células T humanas primárias Naïve e Memória

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Imunologia e Infecção

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Aplicação Clínica de<em&gt; A Bela Adormecida</em&gt; Artificial e Antigen Presenting Cells para modificar geneticamente células T de Sangue do Cordão Umbilical e periférica

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Imagens em tempo real de heterotípica plaquetas neutrófilos Interações sobre o endotélio ativado durante a inflamação vascular e formação de trombos em camundongos vivos

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

O isolamento de células dendríticas mielóides e células epiteliais de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Diferenciação Pan-Myeloid do sangue humano do cabo derivado CD34+ da haste hematopoietic e das pilhas do progenitor

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Biologia do Desenvolvimento

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.

Isolamento de Progenitores de Megacaiócitos de Rato

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

Biologia

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.

This protocol describes in detail all the steps involved in obtaining leukofilter-derived CD34+ hematopoietic progenitors and their in vitro differentiation and maturation into proplatelet-bearing megakaryocytes that are able to release platelets in the culture medium. This procedure is useful for in-depth analysis of cellular and molecular mechanisms controlling megakaryopoiesis.

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Diferenciação e Formação de Plaquetas

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing ...

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.

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.

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 ...

<meta charset="utf8"></head><body>Modelagem de eritropoiese usando HSPCs de sangue de cordão umbilical e medula óssea

A Comprehensive Pipeline to Assess the Efficiency of Human Erythropoiesis In Vitro and Ex Vivo

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 &plusmn; 20 billion cells per day), is crucial for maintaining individual health. Any disruption in this pathway can have significant consequences, leading to health issues. According to the World Health Organization, an estimated 25% of the global population presents symptoms of anemia. This protocol describes how to generate human erythroid cells both in vitro using hematopoietic stem and progenitor cells (HSPCs) from sources such as umbilical cord blood (UCB) or blood taken from healthy donors and ex vivo with HSPCs isolated from patients' bone marrow. Using genetic approach, genes of interest can be modulated in HSPCs, and their impact on erythropoiesis can be monitored at various stages of the differentiation process. This method allows for the screening of compounds perturbing, enhancing, or rescuing the capacity of HSPCs to differentiate into mature erythroid cells and to investigate the role of genes of interest during the erythroid differentiation process.

<meta charset="utf8"></head><body>Autor em destaque: Avançando na pesquisa de eritropoiese - um pipeline simplificado para avaliar a função das células-tronco hematopoiéticas em síndromes mielodisp

Modeling erythropoiesis provides a valuable chance to comprehend the biological significance of actors in this process and to evaluate novel therapeutic approaches that may alleviate differentiation defects. Here, we describe a simple and reliable method to efficiently differentiate CD34+ hematopoietic stem and progenitor cells ex vivo.

Um pipeline abrangente para avaliar a eficiência da eritropoiese humana in vitro e ex vivo

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 &plusmn; 20 ...

Megacariócito diferenciação e formação de plaquetas de humanos cabo hemoderivados CD34 células⁺

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