Damos las gracias a Nobuyuki Onai, Hitoshi Takizawa y Manz Markus de preciosos consejos. Este trabajo fue financiado por la Swiss National Science Foundation, la Liga Suiza contra el Cáncer y la Fundación per la Ricerca Ticinese sul Cancro.

1. Preparación de la suspensión de la célula de la médula ósea <ol><li> Practicar la eutanasia con el ratón y colocar al animal en una sartén inoxidable y el etanol de pulverización 70% sobre su abdomen y la espalda. Recoger fémur y la tibia de las patas traseras, y la columna vertebral. </li><li> Con precisión todos los residuos de los tejidos blandos de la columna vertebral con unas tijeras de punta afilados y de los huesos de las piernas con una gasa. Huesos en tienda a un tubo Falcon de 50 ml con 30 ml de medio RPMI que contenía 10% inactivado por calor suero fetal bovino (FBS), suplementado con 50 mM β-mercaptoetanol, 100 uM MEM no aminoácidos esenciales, 1 mM de piruvato sódico MEM, 2 mM de L-glutamina, 100 ug / ml de estreptomicina y penicilina 100 U / ml. </li><li> Cubrir el fondo de un mortero previamente esterilizado con filtros estériles. </li><li> Transferencia de los huesos en el mortero y los cubren con una capa de filtros con el fin de montar una superficie de fricción. </li><li> Preparar un tubo Falcon de 50 ml provisto de un filtro de Falcon. </li><li> Rompe los huesoscon la ayuda de una mano de mortero hasta obtener una suspensión celular homogénea. </li><li> Transferir la suspensión en el tubo. </li><li> Añadir al mortero 5 ml de medio RPMI completo fresco para lavar y recoger las células restantes. </li><li> Repita los dos últimos puntos hasta que los huesos homogeneizados parece clara. </li><li> Transferir y filtrar en un tubo nuevo la suspensión de células a fin de eliminar los restos de tejido restante. </li><li> Centrifugar el tubo a 1.500 rpm durante 5 minutos y retirar con cuidado el sobrenadante por aspiración. Asegúrese de no perturbar el sedimento celular. </li></ol> 2. El análisis fenotípico <ol><li> Resuspender y lavar las células de ratones individuales en 15 ml de PBS y FBS al 2%. </li><li> Centrifugar el tubo a 1.500 rpm durante 5 minutos y retirar con cuidado el sobrenadante por aspiración. Asegúrese de no perturbar el sedimento. </li><li> Suspender el precipitado en 5 ml de PBS y 2% de FBS y cuentan las células. </li><li> Transferencia de 1 × 10 6 células / pocillo en un fondo de 96 pozos de todo elplato. </li><li> Centrifugar la placa a 1.500 rpm durante 5 min. </li><li> Preparar una mezcla de mAbs dirigidos contra los siguientes marcadores de superficie. Diluir como se indica en PBS y 2% de FBS para teñir las células. <ul type="disc"><li> CD4, CD8, CD3, CD45R, CD19, Gr1, Ter119, NK1.1 conjugados con PE-Cy5 1:200 </li><li> c-Kit conjugado a APC 1:100 </li><li> Conjugado con biotina 1:50 Sca1 </li><li> Conjugado con FITC 1:50 CD34 </li><li> FcγR conjugado a PE 1:100 </li><li> La IL-7R conjugado con PE-Cy7 1:100 </li></ul></li><li> Se desecha el sobrenadante y se añaden 50 l de mezcla mAbs a cada pocillo. Se disocian en células individuales con la pipeta. </li><li> Se incuban las células durante 20 minutos en la oscuridad a 4 ° C. </li><li> Lavar las células dos veces con 150 l de PBS y 2% de FBS. </li><li> Centrifugar la placa a 1.500 rpm durante 5 min y descartar el sobrenadante invirtiendo la placa. </li><li> Diluir estreptavidina en PBS y 2% de FBS, como se indica para teñir las células. <ul type="disc"><li> Estreptavidina conjugadacerrada a APC-Cy7 1:300 </li></ul></li><li> Añadir 50 l de la solución a cada pocillo. Se disocian en suspensión de células individuales con la pipeta. </li><li> Se incuban las células durante 10 minutos en la oscuridad a 4 ° C. </li><li> Lavar las células dos veces con 150 l de PBS y 2% de FBS. </li><li> Centrifugar la placa a 1.500 rpm durante 5 min y descartar el sobrenadante. </li><li> Resuspender las células en 100 l de PBS y FBS al 2%. </li><li> Adquirir muestras en FACS. </li></ol> 3. Enriquecimiento magnética de c-Kit + en las células <ol><li> Preparar una solución tampón que contenía PBS, pH 7,2, 0,5% albúmina de suero bovino (BSA), y 2 mM de EDTA. </li><li> Resuspender el sedimento celular del hueso médulas derivado de al menos 10 ratones en 1 ml de solución tampón que contiene c-Kit conjugado a APC AcMo diluido 1:50. </li><li> Se incuban las células durante 20 minutos en la oscuridad a 4 ° C. </li><li> Lavar dos veces las células con 40 ml de solución tampón. </li><li> Centrifugar el tubo a 1.500 rpm durante 5 minutos y retirar con cuidado tque sobrenadante por aspiración. Asegúrese de no perturbar el sedimento. </li><li> Resuspender pellet de células y se añaden 50 l de anti-APC MicroBeads para cada ratón sacrificados directamente a la pastilla. </li><li> Mezclar bien e incubar durante 20 minutos en la oscuridad a 4 ° C. </li><li> Lavar las células mediante la adición de 40 ml de solución tampón y se centrifuga a 1.500 rpm durante 5 min. Aspirar el sobrenadante por completo. </li><li> Resuspender el sedimento celular en múltiplo de 4 ml de solución tampón cada tres ratones sacrificados. </li><li> MACS Place columnas LS en el campo magnético de un separador MACS adecuado. Utilice una columna de cada tres ratones sacrificados. </li><li> Preparar las columnas de un enjuague con 4 ml de solución tampón. </li><li> Aplicar suspensión celular en las columnas. </li><li> Recoger las células no marcadas que pasan a través y lavar la columna con 4 ml de solución tampón. Realizar pasos de lavado mediante la adición de 4 ml de solución tampón de tres veces. Añadir nuevo buffer cuando el depósito se vacía en la columna. </li><li> Quitar columnas del separador y place cada uno en la parte superior de 15 mL tubos Falcon. </li><li> Pipetear 4 ml de solución tampón en cada columna. Inmediatamente enjuagar las células marcadas magnéticamente, empujando el émbolo en la columna. </li><li> Centrifugar los tubos a 1.500 rpm durante 5 minutos y retirar con cuidado el sobrenadante por aspiración. </li><li> Resuspender cada pellet con 1 ml de solución tampón y pellets de grupo en un solo tubo de 15 ml de halcón. </li></ol> 4. Clasificación de las células madre hematopoyéticas y Lineage Progenitores comprometidos <ol><li> Preparar una mezcla de mAbs dirigidos contra los siguientes marcadores de superficie. Diluir como se indica en 500 l de PBS y FBS al 2% para teñir las células. <ul type="disc"><li> CD4, CD8, CD3, CD45R, CD19, Gr1, Ter119, NK1.1 conjugados con PE-Cy5 1:200 </li><li> c-Kit conjugado a APC 1:100 </li><li> Sca-1 conjugado con biotina 1:50 </li><li> Conjugado con FITC 1:50 CD34 </li><li> FcγR conjugado a PE 1:100 </li></ul></li><li> Centrifugar el tubo de la previous la sección a 1.500 rpm durante 5 minutos y retirar con cuidado el sobrenadante por aspiración. </li><li> Añadir la mezcla de mAb a la pastilla. Se disocian en suspensión de células individuales con la pipeta. </li><li> Se incuban las células durante 20 minutos en la oscuridad a 4 ° C. </li><li> Lavar dos veces las células con 14 ml de PBS y FBS al 2%. </li><li> Centrifugar el tubo a 1.500 rpm durante 5 min y descartar el sobrenadante. </li><li> Diluir estreptavidina como se indica en 500 l de PBS y 2% de FBS para teñir las células. <ul type="disc"><li> Estreptavidina conjugada con APC-Cy7 1:300 </li></ul></li><li> Añadir la solución al tubo. Se disocian en suspensión de células individuales con la pipeta. </li><li> Se incuban las células durante 10 minutos en la oscuridad a 4 ° C. </li><li> Lavar dos veces las células con 14 ml de PBS y 2% de FBS. </li><li> Centrifugar el tubo a 1.500 rpm durante 5 min y descartar el sobrenadante. </li><li> Resuspender las células en 500 l de PBS y FBS al 2%. </li><li> Transferir y filtrar la suspensión de células en un tubo nuevo ml 5 con el fin de Eliminate agregados celulares que pueden interferir con el proceso de selección. </li><li> Añadir 5 l de 7-AAD (utilizarlo diluido 1:100) a la suspensión celular con el fin de excluir 7-AAD + las células muertas de las poblaciones ordenados. </li><li> Clasificar las células con un FACSAria BD de acuerdo con los siguientes fenotipos: <ul type="disc"><li> LKS células: LIN - c-Kit + Sca-1 + </li><li> LT-HSC: LKS CD34 - </li><li> ST-HSC: LKS CD34 + </li><li> BPM: Lin - c-Kit + Sca-1 baja FcγR alta CD34 + </li><li> Diputados al Parlamento Europeo: Lin - c-Kit + Sca-1 baja FcγR baja CD34 - </li><li> CMP: Lin - c-Kit + Sca-1 baja FcγR baja CD34 + </li></ul></li></ol> 5. Los resultados representativos La figura 1 muestra y el ejemplo del procedimiento de puerta electrónica apuntuación progenitores linfoides comunes (CLPS) como Lin - / c-Kit altas / interleuquina (IL)-7R + en las células; Lin - / c-kit + / Sca-1 + (LKS) y Lin - / c-Kit + / Sca -1 LO (c-Kit +) las células, desde el c-Kit + puerta, comunes progenitores mieloides (CMPS) se identifican como FcγR lociones células CD34 +, progenitores de granulocitos / monocitos (BPF), FcγR hi células CD34 + y megacariocitos / progenitores de eritrocitos (los diputados) como FcγR he CD34-células, desde la puerta de LKS, LT y ST-HSC HSC-se identifican como CD34 - y CD34 + en las células, respectivamente. Esta suspensión celular de médula ósea puede ser enriquecido para c-Kit + en las células con perlas magnéticas (Fig. 2); LT-HSC y ST-HSC puede ser clasificado de acuerdo a la expresión CD34, así como otros progenitores de acuerdo con el fenotipo descrito anteriormente. HSC puede ser ana<html> lyzed en microscopía confocal de imágenes en vivo como se muestra en la Figura 4, donde CD34 - células se tiñeron con el compuesto de unión a nucleótidos quinacrina 11, así como nuclear rojo (DRAQ5). En CMH, pero no BPF, quinacrina-positivas vesículas son visibles en el citoplasma 12. Además, las CMH ordenados puede ser utilizado en los experimentos funcionales como se demuestra en la figura 5, que muestra el aumento de Ca 2 + citosólico en LT-HSC por la activación de los receptores purinérgicos P2 a la exposición a la adenosina-trifosfato (ATP) y ionomicina 12. <img alt="Figura 1" src="/files/ftp_upload/3736/3736fig1.jpg" /> Figura 1. Representante de análisis gráfico de puntos de Lin-BM células. Procedimiento de puerta electrónica en citómetro de flujo para identificar y anotar progenitores hematopoyéticos y HSCs de suspensión de células de médula ósea tiñeron como se describe en el texto del protocolo. pload/3736/3736fig2.jpg &quot;/&gt; Figura 2. Un esquema de enriquecimiento selectivo de c-Kit + en las células de la médula ósea. <img alt="Figura 3" src="/files/ftp_upload/3736/3736fig3.jpg" /> Figura 3 Análisis del ciclo celular de la electrónica LKS cerradas CD34 -. CMH en los controles sanos y ratones con EII teñidas con anticuerpos Ki-67 y DAPI. Las células fueron fijadas y permeabilized con Lyse / Fijar y Perm tampones seguido de tinción con FITC conjugado con Ki-67 y DAPI a 1 mg / ml. Ciclo de las células apenas si es perceptible en el LKS CD34 - compartimiento de CMH de los controles sanos 12. <img alt="Figura 4" src="/files/ftp_upload/3736/3736fig4.jpg" /> . Figura 4 en vivo de imágenes de microscopía confocal de la FACS ordenados CD34 - CMH y las BPF manchados con la quinacrina compuesto de nucleótidos vinculante y nucleares rojo (DRAQ5). Los cuadrantes pequeños muestran pos quinacrinavesículas itive detectado en el citoplasma de las CMH, pero BPM no. <img alt="Figura 5" src="/files/ftp_upload/3736/3736fig5.jpg" /> Figura 5. Citosólica de Ca 2 + elevaciones de la CMH ordenados cargados con Fura-2 y estimuladas con ATP y ionomicina. Trazas de células individuales se muestran. Todas las células fueron sensibles a ATP extracelular que muestran un aumento importante en Ca 2 + citosólico tras la adición 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>

No hay conflictos de interés declarado.

El método aquí descrito permite el análisis rápido y preciso de la hematopoyesis en ratones individuales (Figura 1). Este análisis en diversos parámetros experimentales, incluidos los modelos murinos de inflamación, autoinmunidad, inmunodeficiencia, enfermedades degenerativas, trastornos metabólicos y el cáncer, permite abordar el impacto de las condiciones patológicas en la hematopoyesis. La Figura 3 muestra el análisis de la actividad de las células en el ciclo de LKS elec...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> RPMI 1640 </td><td> Gibco </td><td> 42401 </td></tr><tr><td> MEM AANE 100X </td><td> Gibco </td><td> 11140 </td></tr><tr><td> Piruvato de sodio </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> Filtro de la célula 40 micras </td><td> BD Falcon </td><td> 352340 </td></tr><tr><td> 7-AAD Solución colorante </td><td> BD Pharmingen </td><td> 559925 </td></tr><tr><td> Lyse / Fix tampón </td><td> BD Pharmingen </td><td> 558049 </td></tr><tr><td> Perm tampón 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 MicroBeads </td><td> Miltenyi Biotec </td><td> 130-090-855 </td></tr><tr><td> Columnas 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

La médula ósea es el lugar principal donde las CMH y más maduras las células sanguíneas progenitoras de linaje residen y se diferencian en un organismo adulto. CMH constituyen una población de células minuto de células pluripotentes, capaces de generar todos los linajes de células sanguíneas para un tiempo de vida 1. La disección molecular de la homeostasis del CMH en la médula ósea tiene implicaciones importantes en la hematopoyesis, oncología y medicina regenerativa. Se describe el protocolo de etiquetado con anticuerpos fluorescentes y el procedimiento de puerta electrónica en citometría de flujo para anotar subconjuntos progenitoras hematopoyéticas y distribución HSCs en ratones individuales (Fig. 1). Además, se describe un método para enriquecer ampliamente progenitores hematopoyéticos, así como a largo plazo (LT) ya corto plazo (ST) la reconstitución de HSCs agrupados de suspensiones de células de médula ósea por enriquecimiento magnética de las células que expresan c-kit. La preparación de células resultante se puede utilizar para ordenar los subconjuntos seleccionados para in vitro een estudios in vivo funcionales (fig. 2). Tanto los osteoblastos trabeculares 2,3 y 4 endotelio sinusoidal constituyen nichos funcionales de apoyo CMH en la médula ósea. Hay varios mecanismos que en el nicho de los osteoblastos, como un subconjunto de N-cadherina + 3 osteoblastos y la interacción de la Tie2 tirosina quinasa del receptor se expresa en las CMH con su angiopoyetina-1 ligando 5 de acuerdo en la determinación de la quietud CMH. &quot;Hibernación&quot; en la médula ósea es crucial para proteger a las HSCs de replicación y eventual agotamiento en la actividad un ciclo excesivo 6. Estímulos exógenos que actúan sobre las células del sistema inmune innato como ligandos de los receptores Toll-like 7 y el interferón-alfa 6 también se puede inducir la proliferación y diferenciación de las CEH en el linaje de los progenitores comprometidos. Recientemente, una población de HSC de ratón latentes dentro de la lin - c-Kit + Sca-1 + CD150 + CD48 - CD34 - la población ha sido descrito 8. Clasificación de las células CD34 sobre la base de expresión de la suspensión de células hematopoyéticas progenitoras enriquecido como se ha descrito aquí permite el aislamiento de ambos quiescente autorrenovación LT-HSC y ST-CMH 9. Un procedimiento similar basado en el agotamiento de linaje de células positivas y la clasificación de LT-HSC con CD48 y Flk2 anticuerpos ha sido descrita previamente 10. En el presente informe se proporciona un protocolo para la caracterización fenotípica y ex vivo de células análisis del ciclo de progenitores hematopoyéticos, que pueden ser útiles para el seguimiento de la hematopoyesis en diferentes condiciones fisiológicas y patológicas. Además, se describe un FACS sorting procedimiento para CMH, que pueden ser utilizados para definir los factores y mecanismos que regulan su auto-renovación, expansión y diferenciación en la biología celular y ensayos de transducción de señales, así como para el trasplante.

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

Un método para analizar la distribución de progenitores hematopoyéticos de médula ósea en citometría de flujo, así como para aislar eficazmente altamente purificadas células madre hematopoyéticas (HSC) se describe. El procedimiento de aislamiento se basa esencialmente en el enriquecimiento magnética de c-kit + células y células de clasificación para purificar las CMH para los estudios celulares y moleculares.

El análisis fenotípico y aislamiento de células madre hematopoyéticas murinas y progenitores comprometidos Lineage

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

phenotypic-analysis-isolation-murine-hematopoietic-stem-cells-lineage

Research

JoVE Journal

Biology

18.5K vistas.  Bellinzona (Switzerland). Un método para analizar la distribución de progenitores hematopoyéticos de médula ósea en citometría de flujo, así como para aislar eficazmente altamente purificadas células madre hematopoyéticas (HSC) se describe. El procedimiento de aislamiento se basa esencialmente en el enriquecimiento magnética de c-kit + células y células de clasificación para purificar las CMH para los estudios celulares y moleculares.

Video: 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

Aislamiento y trasplante de células madre hematopoyéticas (HSC)

Investigación

Biología

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.

Derivación de células madre hematopoyéticas de células madre embrionarias murinas

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.

Aislamiento y análisis de las células madre hematopoyéticas de la 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.

El aislamiento de las primeras células madre hematopoyéticas murinas saco vitelino y 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.

Lentiviral mediada por derribo Durante Ex Vivo Eritropoyesis de las células estaminales hematopoyéticas

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.

Inducción y Análisis de epitelial a mesenquimal de transición

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;

Aislamiento, cultivo y trasplante 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.

Infección retroviral murino de células madre embrionarias células derivadas embrioide cuerpo para el Análisis de la diferenciación hematopoyética

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.

Identificación de los factores clave que regula la auto-renovación y diferenciación de las células precursoras hematopoyéticas EML por análisis de secuenciación de ARN-

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.

Medición de la masa mitocondrial y el potencial de la membrana en células madre hematopoyéticas y células T por citometría de flujo

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