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 trabalho foi parcialmente apoiado por um Fundo-A-Fellow subvenção da Myotonic Dystrophy Foundation (MDF-FF-2014-0013) eo Cientista Development Grant da American Heart Association (16SDG29320001).

Os ratinhos transgénicos de tipo selvagem e Nestin-GFP foram alojados na instalação animal na Universidade de Minnesota. Todos os procedimentos experimentais foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais da Universidade de Minnesota e estavam de acordo com o Guia do NIH para o Cuidado e Uso de Animais de Laboratório. 1. Dissecção muscular e isolamento de célula única <ol><li> Euthanize camundongos adultos (6-10 semanas, ambos do sexo masculino e feminino) com tribromoetanol (250 mg / kg, ip) e esterilizar a pele do abdômen com etanol 70%. NOTA: O tribromoetanol foi usado aqui em vez de cetamina para anestesia / eutanásia, já que a ketamina é conhecida por interagir com os receptores NMDA, o que poderia potencialmente ter um impacto no estudo. </li><li> Coloque os ratos na posição supina e use um bisturi para fazer uma incisão horizontal na pele abdominal. Descasque a pele com a mão, puxando em direções opostas para expor os músculos dos membros posteriores. </li><li> ColecionarT os músculos de ambos os membros posteriores usando fórceps e tesoura. Armazená-los em solução salina tamponada com fosfato (PBS) estéril suplementada com 1% de penicilina-estreptomicina (P / S) em gelo. </li><li> Lavar os músculos do membro posterior dissecados em PBS gelado suplementado com P / S a 1% duas vezes e transferi-los para uma placa estéril de 10 cm. </li><li> Cuidadosamente dissecar os nervos, vasos sanguíneos e tecido conjuntivo dos músculos usando pinças e tesoura sob um microscópio de dissecção em 2X ampliação. </li><li> Cortar finamente e picar os músculos em pedaços pequenos (1-2 mm 3 ) usando tesouras estéreis e lâminas. Se necessário, adicionar uma pequena quantidade de Dulbecco Modified Eagle Medium (DMEM) para garantir que os músculos não são secos. </li><li> Quebrar mecanicamente o tecido por pipetagem para cima e para baixo através de uma pipeta serológica de 10 ml 10 vezes. </li><li> Adicionar a solução de digestão recentemente preparada (DMEM suplementado com colagenase de tipo 2 a 0,2%) à mistura. Incubar a 37 ° C por 2 h com gentlAgitação a 35 revoluções / min. </li><li> Triturar utilizando uma agulha de 18 G para homogeneizar a mistura. Em seguida, centrifugar a 500 xg durante 5 min. Descartar o sobrenadante e depois ressuspender o sedimento em 0,25% de tripsina / EDTA. Incubar a 37 ° C durante 10 min. Repita o passo de centrifugação mais duas vezes. </li><li> Adicionar 10 mL de DMEM suplementado com 20% de soro fetal bovino (FBS) à solução e centrifugar a 500 xg durante 5 min. Descartar o sobrenadante e ressuspender o sedimento em tampão de lise de células vermelhas do sangue (NH4Cl 155 mM, KHCO3 10 mM e EDTA 0,1 mM). </li><li> Centrifugar a 500 xg durante 5 min. Descartar o sobrenadante e ressuspender o sedimento em tampão de separação (HEPES 20 mM, pH 7,0, EDTA 1 mM e BSA a 1% em PBS livre de Ca / Mg2 + , pH 7,0). Filtrar a mistura através de um filtro de células de 40 um para obter uma suspensão de célula única. </li><li> Centrifugar a 500 xg durante 5 min. Descartar o sobrenadante e ressuspender o sedimento em 1 mL de tampão de separação. </li><li> Conte oNúmero de células com um hemocitômetro e diluir a suspensão de célula única para 5 x 10 6 / mL em tampão de triagem. </li></ol> 2. Coloração e Classificação de Células <ol><li> Preparar os controlos ea amostra conforme descrito na Tabela 1 . Mancha a suspensão de célula única com os respectivos anticorpos em gelo durante 30 min, como descrito na Tabela 1 . </li><li> Centrifugar a 500 xg durante 5 min e lavar os pellets duas vezes com tampão de separação. </li><li> Adicionar DAPI às soluções de célula única, como indicado na Tabela 1. Utilizar 5 μg / mL de DAPI (concentração final) para o controlo de cor única DAPI e 1 μg / mL de DAPI para o controlo de PDGFRβ-PE-FMO e para a amostra. Manter todos os tubos em gelo durante todo o experimento. </li><li> Ative o classificador e o software. Digitalize e insira um chip de classificação de 100 μm quando solicitado. </li><li> Execute a configuração automática ( ie, alinhamento de chip , calibração de gotículas, calibração de fluxo lateral e calibração de atraso de classificaçãoOn) carregando os grânulos de configuração automática quando solicitado. </li><li> Quando a configuração automática estiver concluída, vá para a guia &quot;Experiência&quot;, clique em &quot;Novo&quot; e selecione o &quot;Modelo em Branco&quot; em &quot;Modelos Públicos&quot;. </li><li> Em &quot;Configurações de medição&quot;, digite &quot;DAPI&quot; para &quot;FL1&quot;, &quot;Nestin-GFP&quot; para &quot;FL2&quot; e &quot;PDGFRβ&quot; para &quot;FL3.&quot; Desmarque as caixas de &quot;FL4&quot; - &quot;FL6&quot;. </li><li> Marque as caixas para ativar os lasers 405, 488 e 561 e clique em &quot;Criar Experiência Nova&quot;. </li><li> Selecione a opção &quot;Start Compensation Wizard&quot; e siga as instruções do software &quot;Compensation Wizard&quot; para configurar a compensação. <ol><li> Carregar o controle sem cor e clique em &quot;Iniciar&quot;. Clique em &quot;Detector &amp; Threshold Settings&quot; e ajuste o ganho do sensor dos detectores FSC e BSC para colocar a população na escala. </li><li> de AnúnciosApenas os níveis de ganho dos canais de fluorescência FL1-FL3 para colocar as populações negativas no lado esquerdo dos histogramas. Clique no botão &quot;Gravar&quot; para gravar os dados. </li><li> Carregar controles de uma cor um por um quando solicitado. Clique em &quot;Iniciar e Gravar&quot; para gravar os dados. Ajustar os portões para as populações positivas nos histogramas. Clique em &quot;Avançar&quot;. </li><li> Vá para &quot;Calcular matriz&quot; na guia &quot;Compensação&quot; e clique em &quot;Calcular&quot; no painel &quot;Calcular configurações de compensação&quot; para executar a compensação. Clique em &quot;Concluir&quot; para sair do &quot;Assistente de compensação&quot;. </li></ol></li><li> Carregue o controle PDGFRβ-PE-FMO e clique em &quot;Iniciar&quot;. Desenhe um portão de polígono (Portão A) em torno das células de interesse sob o gráfico &quot;Todos os Eventos&quot;. </li><li> Clique duas vezes dentro do portão A para criar um gráfico filho. Altere o eixo Y para DAPI e desenhe uma porta poligonal (Gate B) em torno de células vivas (DAPI low ). Clique duas vezes em iNside Gate B para criar um enredo filho. Mude o eixo X para FSC-H e o eixo Y para FSC-W e desenhe uma porta poligonal (Gate C) em torno dos singlets para eliminar dupletos. </li><li> Clique duas vezes dentro do Gate C para criar um gráfico filho. Mudar o eixo X para Nestin-GFP e o eixo Y para PDGFRβ-PE. Clique no botão &quot;Gravar&quot; para gravar os dados. </li><li> Carregue a amostra e repita os passos 2.11-2.12. Após a gravação, clique em &quot;Pausar&quot; para preservar a amostra. </li><li> Definir os limites de passagem para as células PDGFRβ + e Nestin-GFP + com base no controlo PDGFRβ-PE-FMO. Draw portões para o PDGFRβ + Nestin-GFP - e PDGFRβ + Nestin-GFP + populações. </li><li> Em &quot;Método de classificação&quot;, selecione &quot;Tubos de 2 vias&quot; e atribua as células &quot;PDGFRβ + Nestin-GFP - &quot; e &quot;PDGFRβ + Nestin-GFP + &quot; aos tubos coletores esquerdo e direitoEspecificamente. Montar os tubos de recolha de 15 mL de tampão de triagem na fase de recolha e clicar no botão &quot;Carregar colecção&quot;. </li><li> Clique no botão &quot;Continuar&quot; para manter a amostra em execução. Clique em &quot;Start Sort&quot; para coletar as células PDGFRβ + Nestin-GFP - (tipo I) e células PDGFRβ + Nestin-GFP + (tipo II pericytes). </li></ol> 3. Análises pós-triagem <ol><li> Centrifugar as células classificadas a 500 xg durante 5 min, ressuspender a pastilha em 1 mL de meio pericito (ver Tabela de Materiais ) e contar a densidade celular utilizando um hemocitómetro. </li><li> Os pericitos tipo I e tipo II de sementes em lamínulas revestidas com poli-D-lisina (PDL) a ~ 1 x 10 4 células / cm2. Crescer em meio pericíto durante 3 dias a 37 ° C com 5% de CO2. </li><li> No dia 3, examinar a morfologia do pericito (sob contraste de fase) e endógena Nestin-GFP expressão utilizando um micro fluorescenteÂmbito (laser de excitação: 488 nm, filtro de excitação: 470/40 nm, e filtro de emissão: 515/30 nm). Tire as imagens com um objetivo de 20X (0,45 NA). </li><li> Substituir o meio pericítico por meio adipogênico (meio basal MSC + suplemento estimulante adipogênico) e miogênico (DMEM + 2% soro de cavalo) para iniciar a diferenciação adipogênica e miogênica, respectivamente, conforme descrito anteriormente 20 . Alterar o meio a cada 2-3 dias. </li><li> Fixar as células no dia 17 (14 dias após a diferenciação adipogénica / miogénica) em paraformaldeído a 4% (PFA) durante 20 min à temperatura ambiente. NOTA: Cuidado, PFA é um carcinógeno. </li><li> Realizar imunocitoquímica contra perilipina (marcador de adipócito) e S-miosina (marcador de miotubo maduro / miofibra), conforme descrito em publicações anteriores 19 , 20 . <ol><li> Lavar as células fixas 3 vezes em PBS durante 10 min à temperatura ambiente. </li><li> Adicionar tampão de bloqueio (PBSSuplementado com soro de burro a 5%, BSA a 3% e Triton X-100 a 0,3%) e incubar à temperatura ambiente durante 1 h. </li><li> Incubar as células com anticorpos anti-perilipina (2 μg / mL) e / ou anti-S-miosina (2 μg / mL) a 4 ° C durante a noite. </li><li> Lavar as células 3 vezes em PBS durante 10 min à temperatura ambiente. </li><li> Incubar as células com anticorpos anti-coelho anti-coelho (4 μg / mL) e / ou Alexa555 anti-rato (4 μg / mL) de burro Alexa 555 à temperatura ambiente durante 1 h. </li><li> Lavar as células 3 vezes em PBS durante 10 min à temperatura ambiente. </li><li> Monte as células imunocoradas com meio de montagem contendo DAPI (ver tabela de materiais). Examine a expressão de perilipina e S-miosina utilizando um microscópio fluorescente (laser de excitação: 543 nm, excitação de 540/45 nm e emissão de 600/50 nm) e tome imagens sob um objetivo 40X (0,60 NA). </li></ol></li></ol>

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Os pericitos são células perivasculares multipotentes 22 , 23 localizadas na superfície abluminal dos capilares 21 , 26 . Nos músculos esqueléticos, os pericitos são capazes de diferenciar ao longo das vias adipogénicas e / ou miogénicas 19 , 20 , 24 . Estudos recentes revelaram duas subpopulações de p...

A distrofia muscular é uma desordem músculo-degenerativa que não tem nenhum tratamento eficaz até agora. O desenvolvimento de terapias que promovem a regeneração de tecidos tem sido sempre de grande interesse. A regeneração e reparação dos tecidos após os danos dependem das células estaminais / células progenitoras residentes 1 . As células satélites são células precursoras miogénicas comprometidas que contribuem para a regeneração muscular 2 , 3 , 4 , 5 , 6 , 7 . O seu uso clínico, no entanto, é dificultado pela sua migração limitada e baixa taxa de sobrevivência após a injecção, bem como pela sua diminuição da capacidade de diferenciação após a amplificação in vitro 8 , 9 , 10 , 11 . Além do satelliTe células, os músculos esqueléticos também contêm muitas outras populações de células com potencial miogénico 12 , 13 , 14 , 15 , 16 , tais como as células intersticiais positivas ao receptor de factor de crescimento derivado de plaquetas beta (PDGFRp). Há evidências que demonstram que as células PDGFRβ + derivadas de músculo são capazes de se diferenciar em células miogênicas e melhorar a patologia e a função muscular 14 , 17 , 18 , 19 , 20 . O PDGFRβ rotula predominantemente os pericitos 21 , que são células perivasculares com pluripotência 22 , 23 . Além de PDGFRβ, muitos outros marcadores, incluindo Neuron-Glial 2 (NG2) e CD146, também são usados ​​para iDentify pericytes 21 . Deve-se notar, no entanto, que nenhum desses marcadores é pericítico-específico [ 21] . Estudos recentes revelaram dois subtipos de pericitos musculares, chamados de tipo I e tipo II, que expressam marcadores moleculares diferentes e exercem funções distintas 19 , 24 , 25 . Bioquimicamente, os pericitos do tipo I são NG2 + Nestin - , enquanto os pericídios do tipo II são NG2 + Nestin + 19 , 24 . Funcionalmente, os pericitos tipo I podem sofrer diferenciação adipogênica, contribuindo para acúmulo de gordura e / ou fibrose, enquanto que os pericitos de tipo II podem se diferenciar ao longo da via miogênica, contribuindo para a regeneração muscular 19 , 24 , 25 . Estes resultados demonstram que: (1) os pericitos de tipo I podem bE alvo no tratamento de transtornos degenerativos gordos / fibrose, e (2) pericitos de tipo II têm grande potencial terapêutico para a distrofia muscular. Outras investigações e caracterização destas populações requerem um protocolo de isolamento que permita a separação dos pericitos tipo I e tipo II com alto grau de pureza. Atualmente, o isolamento de subpopulações pericíticas depende de camundongos duplamente transgênicos NG2-DsRed e Nestin-GFP 19,24. A disponibilidade de ratinhos NG2-DsRed ea qualidade da maioria dos anticorpos NG2 limitam o uso generalizado deste método. Dado que todos os pericitos NG2 + também expressam PDGFRβ nos músculos esqueléticos 19 , 20 , 24 , hipótese de que NG2 pode ser substituído por PDGFRβ para o isolamento de pericitos e suas subpopulações. Este trabalho descreve um protocolo baseado em FACS queUtiliza a coloração PDGFRβ e o sinal Nestin-GFP. Este método é menos exigente para os investigadores porque: (1) não requer o fundo NG2-DsRed e (2) utiliza anticorpos PDGFRβ comercialmente disponíveis, que são bem caracterizados. Além disso, permite o isolamento simultâneo de pericitos tipo I e tipo II com elevada pureza, permitindo uma investigação mais aprofundada da biologia e potencial terapêutico destas subpopulações de pericitos. Após purificação, estas células podem ser cultivadas em cultura, e as suas morfologias podem ser visualizadas. Este trabalho também mostra que os pericitos tipo I e tipo II isolados usando este método, como os purificados a partir de camundongos duplamente transgênicos, são adipogênicos e miogênicos, 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

Pericitos são células multipotentes perivasculares que mostram heterogeneidade em diferentes órgãos ou mesmo dentro do mesmo tecido. Nos músculos esqueléticos, há pelo menos duas subpopulações de pericitos (chamadas de tipo I e tipo II), que expressam diferentes marcadores moleculares e possuem distintas capacidades de diferenciação. Utilizando ratinhos duplamente transgénicos NG2-DsRed e Nestin-GFP, os pericitos de tipo I (NG2-DsRed + Nestin-GFP-) e de tipo II (NG2-DsRed + Nestin-GFP + ) foram isolados com sucesso. Contudo, a disponibilidade destes ratinhos duplamente transgénicos impede o uso generalizado deste método de purificação. Este trabalho descreve um protocolo alternativo que permite o isolamento fácil e simultâneo de pericitos tipo I e tipo II a partir de músculos esqueléticos. Este protocolo utiliza a técnica de triagem de células activada por fluorescência (FACS) e alvos PDGFRβ, em vez de NG2, juntamente com o sinal de Nestin-GFP. Após o isolamento, o tipo I e tyOs pericitos II apresentam morfologias distintas. Adicionalmente, os pericitos de tipo I e de tipo II isolados com este novo método, como os isolados a partir de ratinhos duplamente transgénicos, são adipogénicos e miogénicos, respectivamente. Esses resultados sugerem que este protocolo pode ser utilizado para isolar subpopulações de pericitos dos músculos esqueléticos e, possivelmente, de outros tecidos.

Os parâmetros FACS, incluindo a intensidade do laser e a compensação do canal, são corrigidos com base nos resultados de controles sem cor e controles de cor única. O controlo PDGFRβ-PE-FMO é utilizado para definir o gating para a população de PDGFRβ-PE + ( Figura 1A ). Entre as células PDGFRβ-PE, duas populações que representam as células Nestin-GFP + e Nestin-GFP estão claramente separadas ( Figura...

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 trabalho descreve um protocolo baseado em FACS que permite o isolamento fácil e simultâneo de pericitos tipo I e tipo II dos músculos esqueléticos.

Isolamento de Pericitos Tipo I e Tipo II de Músculos Esqueléticos de Rato

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 visualizações.  University of Minnesota.  Este trabalho descreve um protocolo baseado em FACS que permite o isolamento fácil e simultâneo de pericitos tipo I e tipo II dos músculos esqueléticos. 

Vídeo: 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.

Isolamento e Quantitative Immunocytochemical caracterização de células Miogênica primários e 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 ...

Biologia do Desenvolvimento

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.

Isolamento e diferenciação de Mioblasts primários de explantes do músculo esquelético do rato

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;.

Isolamento de células endoteliais Microvascular do principal músculo esquelético murino

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

Neurociência

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.

Isolamento e Cultura de miofibras individuais e suas células satélites do músculo esquelético

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

Biologia

Isolation, Culture, and Transplantation of Muscle Satellite Cells

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

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

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

Isolamento, cultura e transplante de células musculares satélite

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

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.

Isolamento de artéria pulmonar células musculares lisas de camundongos recém-nascidos

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.

Isolamento e purificação de Pericytes 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).

Isolamento e Cultura de Miofibra único de um modelo murino de distrofia muscular em emery-dreifuss no desenvolvimento pós-natal antecipado

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.

Um método de alta saída para isolar pericites cerebrais do rato

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.

Isolamento de Populações de Células-Tronco Quiescentes de Músculos Esqueléticos Individuais

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