Agradecemos Nobuyuki Onai, Hitoshi Takizawa e Markus Manz para o conselho precioso. Este trabalho foi financiado pela National Science Foundation suíço, suíço Cancer League e Ticinese Fondazione per la Ricerca sul Cancro.

1. Preparação de suspensão de células da medula óssea <ol><li> Euthanize o mouse e colocar o animal em uma panela inoxidável e spray de etanol 70% em seu abdômen e nas costas. Colete fêmur e da tíbia das pernas traseiras e coluna vertebral. </li><li> Precisão remover todos os resíduos de tecidos moles da coluna vertebral com uma tesoura ponta afiada e das pernas ossos com uma gaze. Ossos loja em um tubo Falcon 50 mL com 30 mL de meio RPMI contendo 10% inactivado pelo calor de soro fetal bovino (FBS), suplementado com 50 mM β-mercaptoetanol, 100 iM MEM não-aminoácidos essenciais, 1 mM de piruvato de sódio MEM, 2 mM de L-glutamina, 100 ug / ml de estreptomicina e 100 U / ml de penicilina. </li><li> Cobrir o fundo de uma argamassa previamente esterilizado com filtros estéreis. </li><li> Transfira os ossos na argamassa e cobrir com uma camada de filtros, a fim de montar uma superfície de atrito. </li><li> Prepara-se uma 50 mL tubo Falcon fornecido com um filtro de Falcon. </li><li> Esmagar os ossoscom a ajuda de um pilão até a obtenção de uma suspensão de células homogéneas. </li><li> Transferir a suspensão dentro do tubo. </li><li> Adicionar ao mL argamassa 5 de meio RPMI fresco completo para lavar e colher as células restantes. </li><li> Repita os dois últimos pontos até os ossos aparecem homogeneizadas claro. </li><li> Transferir e filtrar num tubo novo a suspensão de células a fim de eliminar os detritos tecido restante. </li><li> Centrifugar o tubo a 1.500 rpm durante 5 min e cuidadosamente remover o sobrenadante por aspiração. Certifique-se de não perturbar o pellet celular. </li></ol> 2. Análise fenotípica <ol><li> Ressuspender as células e lava-se de ratinhos individuais em 15 mL de PBS e FBS 2%. </li><li> Centrifugar o tubo a 1.500 rpm durante 5 min e cuidadosamente remover o sobrenadante por aspiração. Certifique-se de não perturbar o sedimento. </li><li> Suspender o sedimento em 5 ml de PBS e FBS 2% e contagem de células. </li><li> Transferência de 1 × 10 6 células / poço em um fundo de poços 96 rodadaplaca. </li><li> Centrifugar a placa a 1500 rpm durante 5 min. </li><li> Prepara-se uma mistura de mAbs dirigidos contra os marcadores de superfície que se seguem. Diluir-los, como indicado em PBS e 2% de FBS para corar as células. <ul type="disc"><li> CD4, CD8, CD3, CD45R, CD19, Gr1, Ter119, NK1.1 conjugado com PE-Cy5 1:200 </li><li> c-Kit conjugado a APC 1:100 </li><li> SCA1 conjugado com biotina 1:50 </li><li> CD34 conjugado com FITC 1:50 </li><li> FcγR conjugado com PE 1:100 </li><li> IL-7R conjugado com PE-Cy7 1:100 </li></ul></li><li> Descartar o sobrenadante e adicionar 50 uL de mistura de mAbs a cada poço. Dissociar-se em células individuais por pipetagem. </li><li> Incubar as células durante 20 min no escuro a 4 ° C. </li><li> Lave as células duas vezes com 150 uL de PBS e 2% de FBS. </li><li> Centrifugar a placa a 1500 rpm durante 5 min e descartar o sobrenadante invertendo a placa. </li><li> Diluir estreptavidina em PBS e 2% de FBS como indicado para corar as células. <ul type="disc"><li> Estreptavidina conjugate para APC-Cy7 1:300 </li></ul></li><li> Adicionar 50 uL da solução a cada poço. Dissociar-se em suspensão de células individuais por pipetagem. </li><li> Incubar as células durante 10 min no escuro a 4 ° C. </li><li> Lave as células duas vezes com 150 uL de PBS e 2% de FBS. </li><li> Centrifugar a placa a 1500 rpm durante 5 min e descartar o sobrenadante. </li><li> Ressuspender as células em 100 uL de PBS e FBS 2%. </li><li> Adquirir amostras em FACS. </li></ol> 3. Enriquecimento magnético de Células + c-Kit <ol><li> Preparar uma solução tampão contendo PBS, pH 7,2, 0,5% de albumina de soro bovino (BSA), e 2 mM de EDTA. </li><li> Ressuspender o sedimento de células de medula óssea derivado de, pelo menos, 10 ratinhos em 1 mL de solução tampão contendo c-Kit mAb conjugado a APC diluído 1:50. </li><li> Incubar as células durante 20 min no escuro a 4 ° C. </li><li> Lavar duas vezes as células com 40 mL de solução tampão. </li><li> Centrifugar o tubo a 1.500 rpm durante 5 min e cuidadosamente remover tele sobrenadante por aspiração. Certifique-se de não perturbar o sedimento. </li><li> Ressuspender o sedimento de células e adicionar 50 uL de anti-APC micropérolas para cada rato sacrificados directamente para o sedimento. </li><li> Misturar bem e incubar durante 20 min no escuro a 4 ° C. </li><li> Lave as células por adição de 40 mL de solução tampão e centrifuga-se a 1.500 rpm durante 5 min. Aspirar completamente o sobrenadante. </li><li> Ressuspender o sedimento de células em múltiplo de 4 mL de solução tampão de três em três ratinhos sacrificados. </li><li> Colocar MACS colunas LS no campo magnético de um separador de MACS adequados. Use uma coluna a cada três ratos submetidos à eutanásia. </li><li> Preparar colunas, por lavagem com 4 mL de solução tampão. </li><li> Aplicar suspensão de células para as colunas. </li><li> Recolher células não marcadas que passam através da coluna e lava-se com 4 mL de solução tampão. Realizar passos de lavagem, adicionando 4 ml de solução tampão de três vezes. Adicionar tampão novo quando o reservatório coluna é esvaziada. </li><li> Remover colunas do separador e place cada um em cima de 15 tubos mL Falcon. </li><li> Pipetar 4 mL de solução tampão para cada coluna. Imediatamente lavar as células magneticamente marcadas, empurrando o êmbolo para dentro da coluna. </li><li> Centrifugar os tubos a 1500 rpm durante 5 min e cuidadosamente remover o sobrenadante por aspiração. </li><li> Ressuspender cada pellet com 1 mL de solução tampão e pelotas de grupo em um único tubo de 15 mL Falcon. </li></ol> 4. Seleção de Células-Tronco Hematopoéticas e Lineage Progenitores comprometidos <ol><li> Prepara-se uma mistura de mAbs dirigidos contra os marcadores de superfície que se seguem. Diluir-los, como indicado em 500 uL de PBS e FBS a 2% para corar as células. <ul type="disc"><li> CD4, CD8, CD3, CD45R, CD19, Gr1, Ter119, NK1.1 conjugado com PE-Cy5 1:200 </li><li> c-Kit conjugado a APC 1:100 </li><li> Sca-1 conjugada com biotina 1:50 </li><li> CD34 conjugado com FITC 1:50 </li><li> FcγR conjugado com PE 1:100 </li></ul></li><li> Centrifugar o tubo a partir da previoua secção a 1.500 rpm durante 5 min e cuidadosamente remover o sobrenadante por aspiração. </li><li> Adicionar a mistura de mAbs para o sedimento. Dissociar-se em suspensão de células individuais por pipetagem. </li><li> Incubar as células durante 20 min no escuro a 4 ° C. </li><li> Lavar duas vezes as células com 14 mL de PBS e FBS 2%. </li><li> Centrifugar o tubo a 1.500 rpm durante 5 min e descartar o sobrenadante. </li><li> Diluir estreptavidina, como indicado em 500 uL de PBS e 2% de FBS para corar as células. <ul type="disc"><li> Estreptavidina conjugada com APC-Cy7 1:300 </li></ul></li><li> Adicionar a solução para o tubo. Dissociar-se em suspensão de células individuais por pipetagem. </li><li> Incubar as células durante 10 min no escuro a 4 ° C. </li><li> Lavar duas vezes as células com 14 mL de PBS e 2% de FBS. </li><li> Centrifugar o tubo a 1.500 rpm durante 5 min e descartar o sobrenadante. </li><li> Ressuspender as células em 500 uL de PBS e FBS 2%. </li><li> Transferir e filtrar a suspensão de células em um tubo novo mL 5, a fim de eliminate agregados celulares que podem interferir com o processo de classificação. </li><li> Adicionar 5 uL de 7-AAD (usá-lo diluído 1:100) à suspensão de células a fim de excluir 7-AAD + células mortas das populações ordenados. </li><li> Células de classificação com um FACSAria BD de acordo com os seguintes fenótipos: <ul type="disc"><li> Células LKS: Lin - c-Kit + Sca-1 + </li><li> LT-HSCs: LKS CD34 - </li><li> ST-HSCs: LKS CD34 + </li><li> BPF: lin - c-Kit + Sca-1 FcγR baixa alta CD34 + </li><li> Deputados: lin - c-Kit + Sca-1 FcγR baixo baixo CD34 - </li><li> CMPs: lin - c-Kit + Sca-1 FcγR baixo baixo CD34 + </li></ul></li></ol> 5. Os resultados representativos A Figura 1 mostra e exemplo do procedimento gating electrónico paraA pontuação comuns progenitores linfóides (CLPs) como Lin - / c-Kit Io / interleucina (IL)-7R + de células; Lin - / c-kit + / Sca-1 + (LKS) e Lin - / c-Kit + / Sca -1 Lo (c-kit +) células, a partir do c-Kit + portão, comuns progenitores mielóides (CMPS) são identificadas como FcγR Io células CD34 +, progenitores de granulócitos / monócitos (BPF) como FcγR hi células CD34 + e megacariócitos / progenitores de eritrócitos (MPE) como FcγR eis CD34 - células; a partir do portão LKS, LT-HSC e ST-HSC são identificados como CD34 - e células CD34 +, respectivamente. Esta suspensão de células de medula óssea pode ser enriquecida para células + c-Kit com esferas magnéticas (Fig. 2); LT-HSC e ST-HSC pode então ser classificados de acordo com a expressão de CD34, bem como outras células progenitoras de acordo com o fenótipo descrito acima. HSCs pode ser ana<html> lisados ​​em microscopia confocal de imagem ao vivo, como mostrado na Figura 4, onde CD34 - células foram coradas com o composto de nucleótidos de ligação quinacrina 11, bem como nuclear vermelho (DRAQ5). Em HSCs, mas não BPF, quinacrina-positivas vesículas são visíveis na 12 citoplasma. Além disso, HSCs ordenados pode ser utilizado em experiências funcionais, tal como demonstrado na figura 5, que mostra o aumento em Ca 2 + citosólico em LT-HSC pela activação dos receptores purinérgicos P2 após a exposição a adenosina-trifosfato (ATP) e ionomicina 12. <img alt="A Figura 1" src="/files/ftp_upload/3736/3736fig1.jpg" /> Figura 1. Representante análise gráfico de pontos de Lin-BM células. Procedimento gating electrónico no citómetro de fluxo para identificar e marcar progenitores hematopoiéticos e HSCs de suspensão de células de medula óssea coradas como descrito no texto de protocolo. pload/3736/3736fig2.jpg &quot;/&gt; Figura 2. Um esquema para o enriquecimento selectivo de células + c-kit da medula óssea. <img alt="A Figura 3" src="/files/ftp_upload/3736/3736fig3.jpg" /> Figura 3 análise do ciclo celular de LKS fechados eletronicamente CD34 -. HSCs em controles normais e ratos com IBD corados com o anticorpo Ki-67 e DAPI. As células foram fixadas e permeabilizadas com Lyse / Fix e Perm buffers seguida de coloração com conjugado com FITC Ki-67 e DAPI menos 1 ug / ml. Ciclismo células são mal se em tudo detectável no LKS CD34 - HSCs compartimento de controles saudáveis ​​12. <img alt="A Figura 4" src="/files/ftp_upload/3736/3736fig4.jpg" /> . Figura 4 Viva microscopia confocal da imagem FACS classificadas CD34 - HSCs e BPF coradas com a quinacrina composto nucleotide-binding e vermelho nuclear (DRAQ5). Os quadrantes mostram pequenas quinacrina posvesículas tivo detectada no citoplasma das HSCs mas não GMPs. <img alt="A Figura 5" src="/files/ftp_upload/3736/3736fig5.jpg" /> Figura 5. Citosólica de Ca 2 + elevações na HSCs ordenados carregadas com Fura-2 e estimuladas com ATP e ionomicina. Traços de células individuais são mostrados. Todas as células foram responsivos à ATP extracelular mostrando um aumento proeminente na citosólica de Ca 2 + após a adição de ATP.

<ol>
	<li>Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+units+of+development,+units+of+regeneration,+and+units+in+evolution.">Stem cells: units of development, units of regeneration, and units in evolution.</a> Cell. 100, 157-168 (2000).</li><li>Calvi, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425, 841-846 (2003).</li><li>Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+haematopoietic+stem+cell+niche+and+control+of+the+niche+size.">Identification of the haematopoietic stem cell niche and control of the niche size.</a> Nature. 425, 836-841 (2003).</li><li>Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121, 1109-1121 (2005).</li><li>Arai, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie2/angiopoietin-1+signaling+regulates+hematopoietic+stem+cell+quiescence+in+the+bone+marrow+niche.">Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche.</a> Cell. 118, 149-161 (2004).</li><li>Essers, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFNalpha+activates+dormant+haematopoietic+stem+cells+in+vivo.">IFNalpha activates dormant haematopoietic stem cells in vivo.</a> Nature. 458, 904-908 (2009).</li><li>Nagai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+on+hematopoietic+progenitor+cells+stimulate+innate+immune+system+replenishment.">Toll-like receptors on hematopoietic progenitor cells stimulate innate immune system replenishment.</a> Immunity. 24, 801-812 (2006).</li><li>Wilson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cells+reversibly+switch+from+dormancy+to+self-renewal+during+homeostasis+and+repair.">Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.</a> Cell. 135, 1118-1129 (2008).</li><li>Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+lymphohematopoietic+reconstitution+by+a+single+CD34-low/negative+hematopoietic+stem+cell.">Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.</a> Science. 273, 242-245 (1996).</li><li>Lo Celso, C., Scadden, D., D, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Transplantation+of+Hematopoietic+Stem+Cells+(HSCs.">Isolation and Transplantation of Hematopoietic Stem Cells (HSCs.</a> J. Vis. Exp. (2), e157 (2007).</li><li>Romanello, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine/paracrine+stimulation+of+purinergic+receptors+in+osteoblasts:+contribution+of+vesicular+ATP+release.">Autocrine/paracrine stimulation of purinergic receptors in osteoblasts: contribution of vesicular ATP release.</a> Biochem. Biophys. Res. Commun. 331, 1429-1438 (2005).</li><li>Casati, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+regulation+of+hematopoietic+stem+cell+cycling+activity+by+ATP.">Cell-autonomous regulation of hematopoietic stem cell cycling activity by ATP.</a> Cell Death Differ. 18, 396-404 (2011).</li><li>Bouma, G., Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunological+and+genetic+basis+of+inflammatory+bowel+disease.">The immunological and genetic basis of inflammatory bowel disease.</a> Nat. Rev. Immunol. 3, 521-533 (2003).</li><li>Takizawa, H., Regoes, R. R., Boddupalli, C. S., Bonhoeffer, S., Manz, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+variation+in+cycling+of+hematopoietic+stem+cells+in+steady+state+and+inflammation.">Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.</a> J. Exp. Med. 208, 273-284 (2011).</li></ol>

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O método aqui descrito permite a análise rápida e precisa da hematopoiese em ratinhos individuais (Figura 1). Esta análise em várias configurações experimentais, incluindo modelos murinos de inflamação, auto-imunidade, imunodeficiências, doenças degenerativas, doenças metabólicas e câncer, permite abordar o impacto de condições patológicas na hematopoiese. Figura 3 mostra a análise da atividade de ciclo celular em LKS eletronicamente fechados CD34 -</...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> RPMI 1640 </td><td> Gibco </td><td> 42401 </td></tr><tr><td> MEM NEAA 100X </td><td> Gibco </td><td> 11140 </td></tr><tr><td> Piruvato de sódio </td><td> Gibco </td><td> 11360 </td></tr><tr><td> PenStrep </td><td> Gibco </td><td> 15070 </td></tr><tr><td> PBS </td><td> Gibco </td><td> 20012 </td></tr><tr><td> FBS </td><td> Gibco </td><td> 16000 </td></tr><tr><td> Célula Filtro 40 mM </td><td> BD Falcon </td><td> 352340 </td></tr><tr><td> 7-AAD Solução de coloração </td><td> BD Pharmingen </td><td> 559925 </td></tr><tr><td> Lyse / Fix tampão </td><td> BD Pharmingen </td><td> 558049 </td></tr><tr><td> Perm tampão III </td><td> BD Pharmingen </td><td> 558050 </td> &lt;/ Tr&gt; <tr><td> Ki-67 </td><td> BD Pharmingen </td><td> 556026 </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D21490 </td></tr><tr><td> CD4 (GK1.5) </td><td> eBioscience </td><td> 150041 </td></tr><tr><td> CD8 (53-6,7) </td><td> eBioscience </td><td> 150081 </td></tr><tr><td> CD3 (145-2C11) </td><td> eBioscience </td><td> 150031 </td></tr><tr><td> CD45R (RA3-6B2) </td><td> eBioscience </td><td> 150452 </td></tr><tr><td> CD19 (6D5) </td><td> eBioscience </td><td> 150193 </td></tr><tr><td> Gr1 (RB6-8C5) </td><td> eBioscience </td><td> 155931 </td></tr><tr><td> Tre119 (TER-119) </td><td> eBioscience </td><td> 155921 </td></tr><tr><td> NK-1.1 (PK136) </td><td> eBioscience </td><td> 455941 </td></tr><tr><td> c-Kit (2B8) </td><td> eBioscience </td><td> 171171 </td></tr><tr><td> Sca-1 (D7) </td><td> eBioscicia </td><td> 135981 </td></tr><tr><td> CD34 (RAM34) </td><td> eBioscience </td><td> 110341 </td></tr><tr><td> FcγR (2.4G2) </td><td> eBioscience </td><td> 553145 </td></tr><tr><td> Anti-APC micropérolas </td><td> Miltenyi Biotec </td><td> 130-090-855 </td></tr><tr><td> Colunas LS </td><td> Miltenyi Biotec </td><td> 130-042-401 </td></tr></tbody></table>

phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors

A medula óssea é o principal local onde HSCs e glóbulos mais maduros progenitores da linhagem residem e se diferenciar em um organismo adulto. HSCs constituem uma população de células minutos de células pluripotentes, capazes de gerar todas as linhagens de células do sangue para um tempo de vida-1. A dissecção molecular da homeostase HSCs na medula óssea tem implicações importantes na hematopoiese, oncologia e medicina regenerativa. Nós descrevemos o protocolo marcação com anticorpos fluorescentes e ao processo gating electrónico na citometria de fluxo para marcar subconjuntos progenitoras hematopoiéticas e distribuição HSCs em ratinhos individuais (Fig. 1). Além disso, nós descrevemos um método para enriquecer extensivamente progenitores hematopoiéticos, bem como a longo prazo (LT) e de curto prazo (ST) reconstituindo HSCs a partir do conjunto de osso suspensões de células de medula através de um enriquecimento magnética de células que expressam c-Kit. A preparação de células resultante pode ser usada para classificar os subconjuntos seleccionados para in vitro eOs estudos in vivo funcionais (Fig. 2). Ambos os osteoblastos trabeculares 2,3 e 4 endotélio sinusoidal constituem nichos funcionais de apoio HSCs na medula óssea. Vários mecanismos no nicho osteoblástico, incluindo um subconjunto de osteoblastos + N-caderina 3 e interação do receptor tirosina quinase TIE2 expressa em HSCs com sua angiopoietina-1 ligando cinco concorrem na determinação quiescência HSCs. &quot;Hibernação&quot; da medula óssea é fundamental para proteger HSCs de replicação e eventual exaustão sobre a atividade de ciclismo excessiva 6. Estímulos exógenos que actuam sobre as células do sistema imune inato, tais como ligandos de receptores Toll-like 7 e interferão-a 6 também pode induzir a proliferação e diferenciação de HSCs em linhagem progenitores comprometidos. Recentemente, uma população de HSCs rato latentes dentro do lin - c-Kit + Sca-1 + CD150 + CD48 - CD34 - A população tem sido descrito 8. Triagem de células com base em CD34 expressão a partir da suspensão de células hematopoiéticas progenitoras enriquecido como descrito aqui permite o isolamento de ambos quiescente auto-renovação LT-HSCs e ST-HSCs 9. Um procedimento semelhante com base na depleção da linhagem de células positivas e triagem de LT-HSC com CD48 e Flk2 anticorpos foi previamente descrita 10. No presente relatório, nós fornecemos um protocolo para a caracterização fenotípica e ex vivo análise do ciclo celular de células progenitoras hematopoéticas, que pode ser útil para monitorar a hematopoiese em diferentes condições fisiológicas e patológicas. Além disso, descrevem um procedimento para a triagem FACS HSCs, que podem ser utilizados para definir os factores e mecanismos que regulam a sua auto-renovação, expansão e diferenciação em biologia celular e os ensaios de transdução de sinal, bem como para o transplante.

Watch this Scientific Journal Video about Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors at JoVE.com

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

Um método para analisar a distribuição de células progenitoras hematopoiéticas da medula óssea em citometria de fluxo, bem como para isolar eficazmente altamente purificados de células estaminais hematopoiéticas (HSCs) é descrito. O processo de isolamento é essencialmente baseado no enriquecimento de células magnética c-kit + e classificação celular para purificar HSCs para estudos celulares e moleculares.

Análise fenotípica e Isolamento de Células-Tronco Hematopoéticas Murino e Lineage comprometidos Progenitores

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

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JoVE Journal

Biology

18.5K visualizações.  Bellinzona (Switzerland). Um método para analisar a distribuição de células progenitoras hematopoiéticas da medula óssea em citometria de fluxo, bem como para isolar eficazmente altamente purificados de células estaminais hematopoiéticas (HSCs) é descrito. O processo de isolamento é essencialmente baseado no enriquecimento de células magnética c-kit + e classificação celular para purificar HSCs para estudos celulares e moleculares.

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

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolamento e Transplante de Células-Tronco Hematopoéticas (HSCs)

Biologia

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivação de Células-Tronco Hematopoéticas murino de Células-Tronco Embrionárias

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolamento e Análise de Células-Tronco Hematopoéticas da Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

Isolamento de Células-Tronco Hematopoéticas início de murino saco vitelino e AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentivirus mediada Durante Knockdown Ex Vivo Eritropoese de Células-Tronco Hematopoéticas

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolamento, cultura e transplante de células musculares satélite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Retroviral Infecção murina Embryonic Stem Cell Derived Cells embrioides corpo de Análise da diferenciação hematopoética

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Identificação dos fatores chave que regula a auto-renovação e diferenciação em células precursoras hematopoéticas EML por Análise RNA-seqüenciamento

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

Medição da massa mitocondrial e potencial de membrana em células-tronco hematopoiéticas e células T por citometria flow

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...