Name: isolation of type i and type ii pericytes from mouse skeletal muscles
Uploaded: 2017-05-26T15:00:00.000+00:00
Duration: 10 min 7 s
Description: Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

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>

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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><tbody><tr><td>Cell Sorter</td><td>Sony</td><td>SH800</td><td></td></tr><tr><td>Automatic Setup Beads</td><td>Sony</td><td>LE-B3001</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995</td><td></td></tr><tr><td>Avertin&amp;nbsp;</td><td>Sigma</td><td>T48402</td><td></td></tr><tr><td>Pericyte Growth Medium</td><td>ScienCell</td><td>1201</td><td></td></tr><tr><td>MSC Basal Medium (Mouse)</td><td>Stemcell Technologies</td><td>5501</td><td></td></tr><tr><td>Adipogenic Stimulatory Supplement (Mouse)</td><td>Stemcell Technologies</td><td>5503</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000</td><td></td></tr><tr><td>Horse Serum</td><td>Sigma</td><td>H1270</td><td></td></tr><tr><td>Collagenase Type 2</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>0.25% Trypsin/EDTA&amp;nbsp;</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>PDL</td><td>Sigma</td><td>P6407</td><td></td></tr><tr><td>PDGFR&amp;beta;-PE Antibody</td><td>eBioscience</td><td>12-1402</td><td></td></tr><tr><td>Perilipin Antibody</td><td>Sigma</td><td>P1998</td><td></td></tr><tr><td>S-Myosin Antibody</td><td>DSHB</td><td>MF-20</td><td></td></tr><tr><td>Alexa 555-anti-rabbit antibody&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>A-31572</td><td></td></tr><tr><td>Alexa 555-anti-mouse antibody</td><td>ThermoFisher Scientific</td><td>A-31570</td><td></td></tr><tr><td>Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>DAPI</td><td>ThermoFisher Scientific</td><td>D1306</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2058</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Fisher Scientific</td><td>A661</td><td></td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P184</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190</td><td></td></tr><tr><td>18 G Needles</td><td>BD</td><td>305196</td><td></td></tr><tr><td>10 mL Serological Pipette</td><td>BD</td><td>357551</td><td></td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111</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 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 ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

Aislamiento de las células endoteliales microvasculares primaria murinas músculo esquelético

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Neurociencias

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Aislamiento y cultivo de fibras individuales y sus células satélite del músculo esquelético adulto

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Biología

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 ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

El aislamiento de la arteria pulmonar células musculares lisas de los ratones recién nacidos

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Aislamiento y Purificación de Pericitas Cardíacos Murinos

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Medicina

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Aislamiento de miofiber único y cultura de un modelo murino de distrofia muscular de Emery-Dreifuss en el desarrollo post-Natal temprano

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

Un método de alta salida para aislar los pericytes cerebrales del ratón

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2&#180;-deoxyuridine (EdU) incorporation assay.

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2&#180;-deoxyuridine incorporation assay.

Aislamiento de poblaciones de células madre quiescentes de músculos esqueléticos individuales

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells ...