Este trabajo fue apoyado parcialmente por una beca del Fondo-A-Fellow de la Myotonic Dystrophy Foundation (MDF-FF-2014-0013) y la Beca de Desarrollo Científico de la American Heart Association (16SDG29320001).

Wildtype y Nestin-GFP ratones transgénicos se alojaron en la instalación de animales en la Universidad de Minnesota. Todos los procedimientos experimentales fueron aprobados por el Comité Institucional de Cuidado y Uso de Animales en la Universidad de Minnesota y estaban de acuerdo con la Guía del NIH para el Cuidado y Uso de Animales de Laboratorio. 1. Disección muscular y aislamiento monocelular <ol><li> Euthanize ratones adultos (6-10 semanas, ambos hombres y mujeres) con tribromoetanol (250 mg / kg, ip) y esterilizar su piel abdomen con etanol al 70%. NOTA: Se utilizó tribrometanol en lugar de ketamina para anestesia / eutanasia, ya que se sabe que la ketamina interactúa con los receptores NMDA, lo que podría tener un impacto en el estudio. </li><li> Coloque los ratones en decúbito supino y utilice un bisturí para realizar una incisión horizontal en la piel abdominal. Pele la piel a mano, tirando en direcciones opuestas para exponer los músculos de los miembros posteriores. </li><li> ColeccionarT los músculos de ambos miembros traseros usando fórceps y tijeras. Almacenarlos en solución salina tamponada con fosfato estéril (PBS) suplementada con penicilina-estreptomicina al 1% (P / S) sobre hielo. </li><li> Lave los músculos de la extremidad posterior disecados en PBS enfriado con hielo suplementado con P / S al 1% dos veces y transfiéralos a una placa estéril de 10 cm. </li><li> Cuidadosamente disecar fuera de los nervios, vasos sanguíneos y tejido conectivo de los músculos con pinzas y tijeras bajo un microscopio de disección con un aumento de 2X. </li><li> Picar finamente y picar los músculos en trozos pequeños (1-2 mm 3 ) con tijeras y cuchillas estériles. Si es necesario, agregue una pequeña cantidad de medio Eagle modificado por Dulbecco (DMEM) para asegurar que los músculos no se sequen. </li><li> Romper mecánicamente el tejido pipeteando hacia arriba y hacia abajo a través de una pipeta serológica de 10 ml 10 veces. </li><li> Añadir la solución de digestión recién hecha (DMEM suplementado con colagenasa de tipo 2 al 0,2%) a la mezcla. Incubar a 37 ° C durante 2 h con gentlAgitación a 35 revoluciones / min. </li><li> Triturar usando una aguja de 18 G para homogeneizar la mezcla. A continuación, centrifugar a 500 xg durante 5 min. Desechar el sobrenadante y luego resuspender el sedimento en tripsina / EDTA al 0,25%. Incubar a 37 ° C durante 10 min. Repita el paso de centrifugación dos veces más. </li><li> Añadir 10 ml de DMEM suplementado con 20% de suero bovino fetal (FBS) a la solución y centrifugar a 500 xg durante 5 min. Desechar el sobrenadante y resuspender el sedimento en tampón de lisis de glóbulos rojos (NH4Cl 155 mM, KHCO3 10 mM y EDTA 0,1 mM). </li><li> Centrifugar a 500 xg durante 5 min. Desechar el sobrenadante y resuspender el sedimento en tampón de clasificación (HEPES 20 mM, pH 7,0, EDTA 1 mM y BSA al 1% en PBS libre de Ca / Mg2 + , pH 7,0). Filtrar la mezcla a través de un filtro de células de 40 μm para obtener una suspensión de una sola célula. </li><li> Centrifugar a 500 xg durante 5 min. Desechar el sobrenadante y resuspender el sedimento en 1 mL de tampón de clasificación. </li><li> Cuenta losNúmero de células con un hemocitómetro y diluir la suspensión de una sola célula a 5 x 106 / ml en tampón de clasificación. </li></ol> 2. Coloración y clasificación de células <ol><li> Prepare los controles y la muestra como se describe en la Tabla 1 . Mancha la suspensión de una sola célula con los respectivos anticuerpos en hielo durante 30 min, como se describe en la Tabla 1 . </li><li> Centrifugar a 500 xg durante 5 min y lavar los gránulos dos veces con tampón de clasificación. </li><li> Añadir DAPI a las soluciones monocelulares, como se indica en la Tabla 1. Utilizar 5 μg / mL de DAPI (concentración final) para el control de un solo color de DAPI y 1 μg / mL de DAPI para el control de PDGFRβ-PE-FMO y la muestra. Mantenga todos los tubos en hielo durante todo el experimento. </li><li> Encienda el clasificador y el software. Escanee e inserte un chip de clasificación de 100 μm cuando se le solicite. </li><li> Realice la configuración automática ( es decir, la alineación de las virutas , la calibración de gotas, la calibración de la corriente lateral y el calibración del retardo de clasificaciónOn) cargando las cuentas de configuración automática cuando se le solicite. </li><li> Cuando finalice la configuración automática, vaya a la pestaña &quot;Experimento&quot;, haga clic en &quot;Nuevo&quot; y seleccione &quot;Plantilla en blanco&quot; en &quot;Plantillas públicas&quot;. </li><li> En &quot;Ajustes de medición&quot;, ingrese &quot;DAPI&quot; para &quot;FL1&quot;, &quot;Nestin-GFP&quot; para &quot;FL2&quot; y &quot;PDGFRβ&quot; para &quot;FL3.&quot; Desmarque las casillas de &quot;FL4&quot; - &quot;FL6&quot;. </li><li> Marque las casillas para activar los láseres 405, 488 y 561 y haga clic en &quot;Crear nuevo experimento&quot;. </li><li> Seleccione la opción &quot;Comenzar Compensación Wizard&quot; y siga las instrucciones del software &quot;Compensation Wizard&quot; para configurar la compensación. <ol><li> Cargue el control sin color y haga clic en &quot;Inicio&quot;. Haga clic en &quot;Detector &amp; Threshold Settings&quot; y ajuste la ganancia del sensor de los detectores FSC y BSC para colocar la población en la escala. </li><li> AnuncioSólo los niveles de ganancia de los canales de fluorescencia FL1-FL3 para colocar las poblaciones negativas en el lado izquierdo de los histogramas. Haga clic en el botón &quot;Grabar&quot; para grabar los datos. </li><li> Cargue controles de un solo color uno por uno cuando se le solicite. Haga clic en &quot;Iniciar y grabar&quot; para grabar los datos. Ajustar las puertas para las poblaciones positivas en los histogramas. Haga clic en Siguiente.&quot; </li><li> Vaya a &quot;Calcular matriz&quot; en la pestaña &quot;Compensación&quot; y haga clic en &quot;Calcular&quot; en el panel &quot;Calcular ajustes de compensación&quot; para realizar la compensación. Haga clic en &quot;Finalizar&quot; para salir del &quot;Asistente de Compensación&quot;. </li></ol></li><li> Cargue el control PDGFRβ-PE-FMO y haga clic en &quot;Inicio&quot;. Dibuje una puerta de polígono (Puerta A) alrededor de las celdas de interés bajo la trama &quot;Todos los eventos&quot;. </li><li> Haga doble clic dentro de la puerta A para crear una trama secundaria. Cambie el eje Y a DAPI y dibuje una puerta polígono (Puerta B) alrededor de células vivas (DAPI bajo ). Haga doble clic en iNside Puerta B para crear una trama infantil. Cambiar el eje X a FSC-H y el eje Y a FSC-W y dibujar una puerta de polígono (Puerta C) alrededor de los singlets para eliminar dobletes. </li><li> Haga doble clic dentro de la puerta C para crear una trama secundaria. Cambiar el eje X a Nestin-GFP y el eje Y a PDGFR β-PE. Haga clic en el botón &quot;Grabar&quot; para grabar los datos. </li><li> Cargue la muestra y repita los pasos 2.11-2.12. Después de la grabación, haga clic en &quot;Pausa&quot; para conservar la muestra. </li><li> Definir los límites de gating para PDGFR β + y Nestin-GFP + células basadas en el PDGFR β-PE-FMO control. Dibujar puertas para el PDGFRβ + Nestin-GFP - y PDGFRβ + Nestin-GFP + poblaciones. </li><li> En &quot;Método de clasificación&quot;, seleccione &quot;Tubos de 2 vías&quot; y asigne las células &quot;PDGFRβ + Nestin-GFP-&quot; y &quot;PDGFRβ + Nestin-GFP + &quot; a los tubos colectores izquierdo y derecho.Espectacularmente. Montar los tubos de recogida de 15 mL en la etapa de recogida y hacer clic en el botón &quot;Cargar Colección&quot;. </li><li> Haga clic en el botón &quot;Reanudar&quot; para mantener la muestra funcionando. Haga clic en &quot;Start Sort&quot; para recoger las células PDGFRβ + Nestin-GFP - (tipo I) y PDGFRβ + Nestin-GFP + (tipo II pericytes). </li></ol> 3. Análisis después de la clasificación <ol><li> Centrifugar las células clasificadas a 500 xg durante 5 min, resuspender el gránulo en 1 mL de medio pericítico (véase Tabla de Materiales ) y contar la densidad celular utilizando un hemocitómetro. </li><li> Los pericitos tipo I y tipo II de semillas en cubreobjetos recubiertos de poli-D-lisina (PDL) a ~ 1 x 104 células / cm2. Crecer en medio pericíto durante 3 días a 37 ° C con 5% de CO 2 . </li><li> El día 3, examinar la morfología del pericito (bajo contraste de fase) y endógeno Nestin-GFP expresión utilizando un fluorescente microAlcance (láser de excitación: 488 nm, filtro de excitación: 470/40 nm, y filtro de emisión: 515/30 nm). Tomar imágenes bajo un objetivo 20X (0.45 NA). </li><li> Sustituir el medio pericítico por medio adipogénico (medio basal MSC de ratón + suplemento estimulante adipogénico) y miogénico (DMEM + 2% de suero de caballo) para iniciar diferenciación adipogénica y miogénica, respectivamente, tal como se ha descrito previamente 20 . Cambie el medio cada 2-3 días. </li><li> Se fijan las células al día 17 (14 días después de la diferenciación adipogénica / miogénica) en paraformaldehído al 4% (PFA) durante 20 minutos a temperatura ambiente. NOTA: Precaución, PFA es un carcinógeno. </li><li> Realizar inmunocitoquímica contra la perilipina (marcador de adipocitos) y S-myosin (myotube maduro / marcador de miofibra), como se describe en publicaciones anteriores 19 , 20 . <ol><li> Lavar las células fijas 3 veces en PBS durante 10 min a temperatura ambiente. </li><li> Añadir tampón de bloqueo (PBSSuplementado con suero de burro al 5%, BSA al 3% y Triton X-100 al 0,3%) e incubar a temperatura ambiente durante 1 h. </li><li> Incubar las células con anticuerpos anti-perilipina (2 μg / mL) y / o anti-S-miosina (2 μg / mL) a 4 ° C durante la noche. </li><li> Lavar las células 3 veces en PBS durante 10 min a temperatura ambiente. </li><li> Incubar las células con anticuerpos Anti-conejo Alexa 555 (4 μg / mL) y / o Alexa555 anti-ratón (4 μg / mL) a temperatura ambiente durante 1 h. </li><li> Lavar las células 3 veces en PBS durante 10 min a temperatura ambiente. </li><li> Montar las células inmunoestimuladas con medio de montaje que contenga DAPI (ver tabla de materiales). Examinar la expresión de perilipina y S-miosina usando un microscopio fluorescente (láser de excitación: 543 nm, excitación de 540/45 nm y emisión de 600/50 nm) y tomar imágenes bajo un objetivo 40X (0.60 NA). </li></ol></li></ol>

<ol>
	<li>Rennert, R. C., Sorkin, M., Garg, R. K., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+recruitment+after+injury:+lessons+for+regenerative+medicine.">Stem cell recruitment after injury: lessons for regenerative medicine.</a> Regenerative medicine. 7 (6), 833-850 (2012).</li><li>Sambasivan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax7-expressing+satellite+cells+are+indispensable+for+adult+skeletal+muscle+regeneration.">Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration.</a> Development. 138 (17), 3647-3656 (2011).</li><li>Relaix, F., Zammit, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells+are+essential+for+skeletal+muscle+regeneration:+the+cell+on+the+edge+returns+centre+stage.">Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage.</a> Development. 139 (16), 2845-2856 (2012).</li><li>von Maltzahn, J., Jones, A. E., Parks, R. J., Rudnicki, 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=Pax7+is+critical+for+the+normal+function+of+satellite+cells+in+adult+skeletal+muscle.">Pax7 is critical for the normal function of satellite cells in adult skeletal muscle.</a> Proc Natl Acad Sci U S A. 110 (41), 16474-16479 (2013).</li><li>Lepper, C., Partridge, T. A., Fan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+absolute+requirement+for+Pax7-positive+satellite+cells+in+acute+injury-induced+skeletal+muscle+regeneration.">An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration.</a> Development. 138 (17), 3639-3646 (2011).</li><li>Kuang, S., Charge, S. B., Seale, P., Huh, M., Rudnicki, 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=Distinct+roles+for+Pax7+and+Pax3+in+adult+regenerative+myogenesis.">Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis.</a> J Cell Biol. 172 (1), 103-113 (2006).</li><li>Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A., Kardon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells,+connective+tissue+fibroblasts+and+their+interactions+are+crucial+for+muscle+regeneration.">Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration.</a> Development. 138 (17), 3625-3637 (2011).</li><li>Morgan, J. E., Pagel, C. N., Sherratt, T., Partridge, T. A. <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+persistence+and+migration+of+myogenic+cells+injected+into+pre-irradiated+muscles+of+mdx+mice.">Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice.</a> J Neurol Sci. 115 (2), 191-200 (1993).</li><li>Beauchamp, J. R., Morgan, J. E., Pagel, C. N., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+myoblast+transplantation+reveal+a+discrete+minority+of+precursors+with+stem+cell-like+properties+as+the+myogenic+source.">Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.</a> J Cell Biol. 144 (6), 1113-1122 (1999).</li><li>Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invited+review:+myoblast+transfer:+a+possible+therapy+for+inherited+myopathies.">Invited review: myoblast transfer: a possible therapy for inherited myopathies.</a> Muscle Nerve. 14 (3), 197-212 (1991).</li><li>Montarras, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309 (5743), 2064-2067 (2005).</li><li>Asakura, A., Seale, P., Girgis-Gabardo, A., Rudnicki, 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=Myogenic+specification+of+side+population+cells+in+skeletal+muscle.">Myogenic specification of side population cells in skeletal muscle.</a> J Cell Biol. 159 (1), 123-134 (2002).</li><li>Tamaki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle-derived+CD34+/45-+and+CD34-/45-+stem+cells+are+situated+hierarchically+upstream+of+Pax7++cells.">Skeletal muscle-derived CD34+/45- and CD34-/45- stem cells are situated hierarchically upstream of Pax7+ cells.</a> Stem Cells Dev. 17 (4), 653-667 (2008).</li><li>Dellavalle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+resident+in+postnatal+skeletal+muscle+differentiate+into+muscle+fibres+and+generate+satellite+cells.">Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells.</a> Nat Commun. 2, 499 (2011).</li><li>Mitchell, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+non-satellite+cell+muscle+resident+progenitor+during+postnatal+development.">Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development.</a> Nat Cell Biol. 12 (3), 257-266 (2010).</li><li>Pannerec, A., Formicola, L., Besson, V., Marazzi, G., Sassoon, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+skeletal+muscle+resident+progenitors+and+their+cell+fate+potentials.">Defining skeletal muscle resident progenitors and their cell fate potentials.</a> Development. 140 (14), 2879-2891 (2013).</li><li>Dellavalle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+of+human+skeletal+muscle+are+myogenic+precursors+distinct+from+satellite+cells.">Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.</a> Nat Cell Biol. 9 (3), 255-267 (2007).</li><li>Kostallari, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+in+the+myovascular+niche+promote+post-natal+myofiber+growth+and+satellite+cell+quiescence.">Pericytes in the myovascular niche promote post-natal myofiber growth and satellite cell quiescence.</a> Development. 142 (7), 1242-1253 (2015).</li><li>Birbrair, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+pericytes+in+skeletal+muscle+regeneration+and+fat+accumulation.">Role of pericytes in skeletal muscle regeneration and fat accumulation.</a> Stem Cells Dev. 22 (16), 2298-2314 (2013).</li><li>Yao, Y., Norris, E. H., Mason, C. E., Strickland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+regulates+PDGFRbeta(+)+cell+stemness+and+muscle+development.">Laminin regulates PDGFRbeta(+) cell stemness and muscle development.</a> Nat Commun. 7, 11415 (2016).</li><li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+developmental,+physiological,+and+pathological+perspectives,+problems,+and+promises.">Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.</a> Dev Cell. 21 (2), 193-215 (2011).</li><li>Dore-Duffy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+pluripotent+cells+of+the+blood+brain+barrier.">Pericytes: pluripotent cells of the blood brain barrier.</a> Curr Pharm Des. 14 (16), 1581-1593 (2008).</li><li>Dore-Duffy, P., Katychev, A., Wang, X., Van Buren, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+microvascular+pericytes+exhibit+multipotential+stem+cell+activity.">CNS microvascular pericytes exhibit multipotential stem cell activity.</a> J Cereb Blood Flow Metab. 26 (5), 613-624 (2006).</li><li>Birbrair, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+pericyte+subtypes+differ+in+their+differentiation+potential.">Skeletal muscle pericyte subtypes differ in their differentiation potential.</a> Stem Cell Res. 10 (1), 67-84 (2013).</li><li>Gautam, J., Nirwane, A., Yao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+differentially+regulates+the+stemness+of+type+I+and+type+II+pericytes.">Laminin differentially regulates the stemness of type I and type II pericytes.</a> Stem Cell Research &amp; Therapy. 8 (1), 28 (2017).</li><li>Birbrair, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+multitasking+cells+in+the+regeneration+of+injured,+diseased,+and+aged+skeletal+muscle.">Pericytes: multitasking cells in the regeneration of injured, diseased, and aged skeletal muscle.</a> Front Aging Neurosci. 6, 245 (2014).</li></ol>

Todos los autores no tienen ningún conflicto de interés para divulgar.

Los pericitos son células perivasculares multipotentes 22 , 23 localizadas en la superficie abluminal de los capilares 21 , 26 . En los músculos esqueléticos, los pericitos son capaces de diferenciarse a lo largo de las vías adipogénicas y / o miogénicas 19 , 20 , 24 . Estudios recientes revelaron dos subp...

La distrofia muscular es un trastorno músculo-degenerativo que no tiene tratamientos efectivos hasta el momento. El desarrollo de terapias que promueven la regeneración tisular siempre ha sido de gran interés. La regeneración y reparación de los tejidos después del daño dependen de las células madre / células progenitoras residentes 1 . Las células satélite son células precursoras miogénicas comprometidas que contribuyen a la regeneración muscular 2 , 3 , 4 , 5 , 6 , 7 . Su uso clínico, sin embargo, se ve obstaculizado por su limitada migración y baja tasa de supervivencia después de la inyección, así como por su disminución de la capacidad de diferenciación después de la amplificación in vitro [ 8 , 9 , 10 , 11] . Además de satelliLos músculos esqueléticos también contienen muchas otras poblaciones celulares con potencial miogénico 12 , 13 , 14 , 15 , 16 , tales como las células intersticiales positivas al receptor de factor de crecimiento derivado de plaquetas beta (PDGFRβ). Existen pruebas que demuestran que las células PDGFRβ + derivadas de los músculos son capaces de diferenciarse en células miogénicas y de mejorar la patología y la función muscular 14 , 17 , 18 , 19 , 20 . PDGFR β predominantemente etiquetas pericitas 21 , que son las células perivasculares con pluripotencia [ 22 , 23] . Además de PDGFRβ, muchos otros marcadores, incluyendo Neuron-Glial 2 (NG2) y CD146, también se utilizan para iDentify pericytes 21 . Debe tenerse en cuenta, sin embargo, que ninguno de estos marcadores es pericítico-específico [ 21] . Estudios recientes revelaron dos subtipos de pericitos musculares, denominados tipo I y tipo II, que expresan diferentes marcadores moleculares y realizan funciones distintas 19 , 24 , 25 . Bioquímicamente, los pericitos de tipo I son NG2 + Nestin - , mientras que los pericitos de tipo II son NG2 + Nestin + 19 , 24 . Funcionalmente, los pericitos tipo I pueden experimentar diferenciación adipogénica, contribuyendo a la acumulación de grasa y / o fibrosis, mientras que los pericitos de tipo II pueden diferenciarse a lo largo de la vía miogénica, contribuyendo a la regeneración muscular 19 , 24 , 25 . Estos resultados demuestran que: (1) los pericitos de tipo I pueden bE dirigidos al tratamiento de trastornos degenerativos grasos / fibrosis, y (2) los pericitos tipo II tienen un gran potencial terapéutico para la distrofia muscular. Una mayor investigación y caracterización de estas poblaciones requiere un protocolo de aislamiento que permita la separación de los pericitos de tipo I y II con un alto nivel de pureza. Actualmente, el aislamiento de las subpoblaciones de pericitos se basa en NG2-DsRed y Nestin-GFP doble-transgénicos ratones [ 19 , 24] . La disponibilidad de ratones NG2-DsRed y la calidad de la mayoría de los anticuerpos NG2 limitan el uso generalizado de este método. Dado que todos los pericitos NG2 + también expresan PDGFRβ en los músculos esqueléticos 19 , 20 , 24 , la hipótesis de que NG2 puede ser sustituido por PDGFR β para el aislamiento de los pericitos y sus subpoblaciones. Este trabajo describe un protocolo basado en FACS queUtiliza tinción PDGFRβ y la señal Nestin-GFP. Este método es menos exigente para los investigadores porque: (1) no requiere el fondo NG2-DsRed y (2) utiliza anticuerpos PDGFRβ comercialmente disponibles, que están bien caracterizados. Además, permite el aislamiento simultáneo de pericitos de tipo I y II con alta pureza, lo que permite investigar más a fondo la biología y el potencial terapéutico de estas subpoblaciones de pericitos. Después de la purificación, estas células se pueden cultivar en cultivo, y sus morfologías pueden visualizarse. Este trabajo también muestra que los pericitos tipo I y tipo II aislados utilizando este método, como los purificados a partir de ratones doblemente transgénicos, son adipogénicos y miogénicos, respectivamente.

<table border="0" cellpadding="0" cellspacing="0" width="705">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Cell Sorter</td>
			<td style="width:173px;">Sony</td>
			<td style="width:133px;">SH800</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Automatic Setup Beads</td>
			<td style="width:173px;">Sony</td>
			<td style="width:133px;">LE-B3001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">DMEM</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">11995</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Avertin&#160;</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:133px;">T48402</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Pericyte Growth Medium</td>
			<td style="width:173px;">ScienCell</td>
			<td style="width:133px;">1201</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">MSC Basal Medium (Mouse)</td>
			<td style="width:173px;">Stemcell Technologies</td>
			<td style="width:133px;">5501</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Adipogenic Stimulatory Supplement (Mouse)</td>
			<td style="width:173px;">Stemcell Technologies</td>
			<td style="width:133px;">5503</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Fetal Bovine Serum</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">16000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Horse Serum</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:133px;">H1270</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Collagenase Type 2</td>
			<td style="width:173px;">Worthington</td>
			<td style="width:133px;">LS004176</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">0.25% Trypsin/EDTA&#160;</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">25200</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Penicillin/Streptomycin</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">15140</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">PDL</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:133px;">P6407</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">PDGFR&#946;-PE Antibody</td>
			<td style="width:173px;">eBioscience</td>
			<td style="width:133px;">12-1402</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Perilipin Antibody</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:133px;">P1998</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">S-Myosin Antibody</td>
			<td style="width:173px;">DSHB</td>
			<td style="width:133px;">MF-20</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Alexa 555-anti-rabbit antibody&#160;</td>
			<td style="width:173px;">ThermoFisher Scientific</td>
			<td style="width:133px;">A-31572</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Alexa 555-anti-mouse antibody</td>
			<td style="width:173px;">ThermoFisher Scientific</td>
			<td style="width:133px;">A-31570</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Mounting Medium with DAPI</td>
			<td style="width:173px;">Vector Laboratories</td>
			<td style="width:133px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">DAPI</td>
			<td style="width:173px;">ThermoFisher Scientific</td>
			<td style="width:133px;">D1306</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">HEPES</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">15630</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">EDTA</td>
			<td style="width:173px;">Fisher</td>
			<td style="width:133px;">BP120</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">BSA</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:133px;">A2058</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:311px;">NH4Cl</td>
			<td style="width:173px;">Fisher Scientific</td>
			<td style="width:133px;">A661</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:311px;">KHCO3</td>
			<td style="width:173px;">Fisher Scientific</td>
			<td style="width:133px;">P184</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">PBS</td>
			<td style="width:173px;">Gibco</td>
			<td style="width:133px;">14190</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">18G Needles</td>
			<td style="width:173px;">BD</td>
			<td style="width:133px;">305196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">10ml Serological Pipette</td>
			<td style="width:173px;">BD</td>
			<td style="width:133px;">357551</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of type i and type ii pericytes from mouse skeletal muscles

Los pericitos son células multipotentes perivasculares que muestran heterogeneidad en diferentes órganos o incluso dentro del mismo tejido. En los músculos esqueléticos, existen al menos dos subpoblaciones pericíticas (llamadas tipo I y tipo II), que expresan diferentes marcadores moleculares y tienen distintas capacidades de diferenciación. Utilizando ratones doblemente transgénicos NG2-DsRed y Nestin-GFP, se han aislado con éxito los pericitos de tipo I (NG2-DsRed + Nestin-GFP-) y de tipo II (NG2-DsRed + Nestin-GFP + ). Sin embargo, la disponibilidad de estos ratones doblemente transgénicos impide el uso generalizado de este método de purificación. Este trabajo describe un protocolo alternativo que permite el aislamiento fácil y simultáneo de los pericitos tipo I y tipo II de los músculos esqueléticos. Este protocolo utiliza la técnica de clasificación de células activada por fluorescencia (FACS) y objetivos PDGFR β, en lugar de NG2, junto con la señal de Nestin-GFP. Después del aislamiento, el tipo I y tyLos pericitos pe II muestran morfologías distintas. Además, los pericitos tipo I y II aislados con este nuevo método, como los aislados de los ratones doblemente transgénicos, son adipogénicos y miogénicos, respectivamente. Estos resultados sugieren que este protocolo puede usarse para aislar las subpoblaciones pericíticas de los músculos esqueléticos y posiblemente de otros tejidos.

Los parámetros FACS, incluyendo la intensidad del láser y la compensación del canal, se corrigen en base a los resultados del control sin color y de los controles de un solo color. El control PDGFR beta - PE - FMO se usa para establecer el gating para la población PDGFR beta - PE + ( Figura 1A ). Entre las células PDGFR β-PE, dos poblaciones que representan Nestin-GFP + y Nestin-GFP - células están claramente separa...

Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Este trabajo describe un protocolo basado en FACS que permite el aislamiento fácil y simultáneo de pericitos tipo I y tipo II de los músculos esqueléticos.

Aislamiento de Pericitos Tipo I y Tipo II de Músculos Esqueléticos de Ratón

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

isolation-of-type-i-and-type-ii-pericytes-from-mouse-skeletal-muscles

Research

JoVE Journal

Developmental Biology

9.1K vistas.  University of Minnesota.  Este trabajo describe un protocolo basado en FACS que permite el aislamiento fácil y simultáneo de pericitos tipo I y tipo II de los músculos esqueléticos. 

Video: Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

Aislamiento y Caracterización cuantitativa inmunocitoquímica de células miogénicas Primarias y fibroblastos de músculo esquelético humano

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Investigación

Biología del Desarrollo

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Aislamiento y caracterización de las células satélite de Músculos Rata Head Branchiomeric

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Uso de mitocondrias aisladas a partir de cantidades mínimas de ratón músculo esquelético para alta microplacas rendimiento Mediciones respiratorias

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

El aislamiento efectivo de los islotes funcionales de páncreas de ratón neonatales

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

El aislamiento y enriquecimiento de hígado progenitoras subconjuntos identificados por una combinación de marcadores de superficie Novel

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

Generación de organoides Cerebral prosencéfalo-tipo estandarizadas y reproducibles de humanos inducida por células madre pluripotentes

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

Disección de la Retina Pupal de Drosophila para inmunohistoquímica, Western análisis y aislamiento de ARN

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

Aislamiento, cultivo, caracterización y diferenciación de células progenitoras musculares humanas del procedimiento de biopsia muscular esquelética

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Aislamiento y diferenciación de los mioblastos primarios de los explantes musculares esqueléticos de ratón

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

Traducción de la purificación de afinidad de ribosoma (TRAP) para investigar el desarrollo de la raíz de Arabidopsis thaliana a una escala específica de tipo de célula

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...