Name: in vitro differentiation of mature myofibers for live imaging
Uploaded: 2017-01-07T19:00:00.000+00:00
Duration: 8 min 12 s
Description: Watch this Scientific Journal Video about In Vitro Differentiation of Mature Myofibers for Live Imaging at JoVE.com

This work was supported by the European Research Council (ERG) and EMBO installation (ERG) and by a PhD fellowship from the Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (MP).

NOTA: Um rato produz mioblastos suficientes para cerca de dois pratos de 35 mm ou dois pratos de geração de imagens ao vivo, então mattings plano, dissecção, e revestimento (passo 2.6) em conformidade. Desde mioblastos são isolados a partir de centrifugações sequenciais e preplating, o protocolo deve ser feito em lotes de 5 - 10 animais. Todos os procedimentos que envolvem indivíduos animais foram aprovados pelo Comitê de Ética Animal do Instituto de Medicina Molecular e da Universidade Pierre et Marie Curie 1. Dissecção do Ratos Neonatal Músculos Hind-membros <ol><li> Preparar todas as soluções previamente (Tabela Materiais) e esterilizar por filtração (filtro de 0,22 nm). Certifique-se de todos os meios de comunicação estão a 37 ° C antes da adição às células, com excepção das formulações contendo matriz de membrana basal (por exemplo, Matrigel). </li><li> Esterilizar o material de dissecção (um de cada: tesoura curva, tesoura reta, pinças regulares,e uma pinça de ponta fina) e bancada de trabalho, limpando-os com etanol 70%. </li><li> Preparar um prato de 100 milímetros de Petri com 5 mL de fosfato de Dulbecco tamponado com solução salina (DPBS) para a recolha de músculo e mantê-lo em gelo até que o passo de trituração. </li><li> Decapitar P6 - ratos P8 com tesoura reta e esterilizar a pele com etanol 70%. </li><li> Faça uma incisão na pele para trás e puxe-o suavemente para os membros posteriores até que seja removido, expondo completamente a musculatura posterior-membro. </li><li> Utilizar as pinças para remover o tecido de gordura, sem danificar os músculos. </li><li> Para remover os músculos posteriores-membros dorsais, manter o membro alongado e dobrar a pata para expor os tendões do salto. Use as tesouras curvadas para músculo separado do osso, a partir dos tendões, deslizando suavemente e de corte para cima. Extirpar os músculos e colocá-los em DPBS gelado. </li><li> Isolar os quadríceps, apertando o músculo com uma pinça de ponta fina e corte em torno dele sem danificar o fêmur ou a articulação do joelho.</li><li> Após dissecação todos os animais, a uma capa de cultura de célula de fluxo laminar estéril, em que todos os passos que se seguem devem ser realizados. </li></ol> Isolamento 2. mioblasto <ol><li> Remover o excesso de DPBS. Picar o tecido com tesoura curva esterilizadas de modo a obter uma massa uniforme. </li><li> Recolhe-se o tecido moído num tubo de centrífuga cónico de 50 ml com 5 ml de mistura de digestão e incubar-a com agitação a 37 ° C durante 90 min. </li><li> Parar a digestão através da adição de 6 ml de meio de dissecção e centrifugar a suspensão durante 5 minutos a 75 xg para sedimentar as restante tecido. </li><li> Recolher cuidadosamente o sobrenadante. Certifique-se de não recolher os restos de tecido. Centrifugar-lo a 350 xg durante 5 min; ressuspender-o em 5 mL de meio de dissecção. </li><li> Filtra-se a suspensão de células através de um coador de células de 40 ^ m. Adicionar 25 mL de meio de dissecção e preplate-lo em um prato de 150 mm para os 4 h numa incubadora de cultura de células (37 ° C e 5% de CO 2 &lt;/ Sub&gt;) para permitir que os fibroblastos para aderir. </li><li> Enquanto preplating, pratos de revestimento com 500 mL de matriz de membrana basal diluído 1: 100 em IMDM a frio durante 1 h, à RT. Lavar uma vez com DPBS e placa as células imediatamente (passo 2.8) ou deixar com meio de crescimento até o chapeamento. </li><li> Após preplating, recolher o sobrenadante e centrifuga-se que a 350 xg durante 10 min. </li><li> Ressuspender-lo no meio de crescimento e contar as células em um hemocitómetro. Ajustar o volume para que entre 150.000 e 250.000 células são banhados por membrana basal prato revestido de matriz. Manter as células numa incubadora de cultura de células. </li></ol> 3. Diferenciação de miofibras NOTA: Depois de 3 d, as células devem começar a fundir e formar miotubos em cerca de 70% de confluência (Figura 1B). <ol><li> Neste ponto, transfectar as células, se desejado, com um siRNA ou de ADN de interesse. Se as células não estão a ser transfectada, mudar directamente para meio de diferenciação e de esquip para o passo 3.4. </li><li> Transfectar com reagentes de transfecção seguindo as instruções do fabricante. Incubam-se as células durante 5 h com complexos de siRNA-lípido (20 nM + 1 ul de reagente) ou complexos ADN-lípido (1 ug + 1 uL de reagente). Otimizar as concentrações de siRNA e de DNA, se necessário. </li><li> Lavá-los uma vez com meio de diferenciação e, em seguida, mudar para o novo meio de diferenciação. </li><li> No dia seguinte, dilui-se a matriz de membrana basal 1: 2 em meio de diferenciação gelada. Remover o meio e adicionar 200 existente ul de matriz arrefecido com gelo para cada prato. </li><li> Incubar durante 30 min numa incubadora de cultura de células. </li><li> Suplementar o meio de diferenciação com agrina (100 ng / mL) e adicionar cuidadosamente 2 mL para as células. </li><li> Cuidadosamente mudar metade do meio a cada 2 d, sempre que completa com agrina a uma concentração final de 100 ng / mL. </li><li> Monitorar diferenciação celular e viabilidade. Dependendo de uma variedade de factores tais como (FBS e Chicken extracto de embrião origens), as células podem ter entre 5-10 diferenciação d para atingir plena maturação (Figura 2). </li></ol> 4. A imunocoloração em pratos de vidro de fundo <ol><li> Para a imunocoloração, em qualquer ponto de tempo de interesse, lavagem das células uma vez com DPBS e corrigi-los com PFA a 4%, à TA, durante 10 min. </li><li> Lave-os duas vezes com DPBS. Neste ponto, as células podem ser armazenadas a 4 ° C. </li><li> Permeabilizar-os com 0,5% de Triton X-100 durante 5 min à TA. </li><li> Lavar duas vezes com PBS e bloquear com solução de bloqueio durante 30 minutos à TA. </li><li> Incubar os com anticorpo primário diluído em solução bloqueadora O / N a 4 ° C. </li><li> Lavar 3x com DPBS durante 5 min à TA. </li><li> Incubar-los com o anticorpo secundário e 0,2 ug / ml de DAPI, durante 1 h, à RT. </li><li> Lavar 3x com DPBS durante 5 min à TA. </li><li> Adicionar 200 mL de meio de montagem e proceder à imagem. </li></ol>

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Os autores não têm nada para revelar.

A utilização deste protocolo para a cultura de mioblastos primários dá origem a um nicho especial que alimenta grandemente o desenvolvimento de miofibras. Isto é parcialmente devido a outros tipos de células que estão também presentes em números muito pequenos. Um equilíbrio entre a concentração de mioblastos e pureza da cultura deve ser alcançado. Uma cultura de células boa também depende da qualidade dos produtos utilizados para a formulação de meio. Todos os produtos derivados de origem animal deve ser cuidadosamente testado. Em nossa experiência, as condições de digestão também devem ser monitorados. Como é habitual para culturas primárias, variabilidade experimental pode ser maior do que em estudos com fibras isoladas imortalizadas ou mioblastos. Esta variabilidade pode ser diminuída pela normalização dos componentes do meio e da digestão, ratos idade e o tamanho, e os pontos de tempo para a manipulação de cultura e recolha de resultados. No entanto, a vantagem de analise em tempo real, os mecanismos complicadosnecessárias para o desenvolvimento de células musculares ultrapassa grandemente a desvantagem da variabilidade. Este protocolo confere as vantagens de abordagens in vitro sem comprometer a diferenciação celular. Miofibras maduras até tríades são formados e as contracções são acoplados a faíscas de cálcio. Estas saídas funcionais podem ser acessados ​​em diferentes condições experimentais. Além disso, pode haver muitas variações técnicas introduzidas no protocolo. Os mioblastos podem ser colhidas a partir de ratos neonatais com mutações de interesse relativamente ao desenvolvimento muscular. As células podem ser lisadas para análise bioquímica em diferentes pontos no tempo de diferenciação. indicadores de cálcio pode ser adicionado à cultura de seguir a sua dinâmica. construções optogenética pode ser usada para impor certas vias de sinalização ou para induzir respostas locais específicos. Finalmente, as miofibras podem ser co-cultivadas com outros tipos de células para estudar as suas interacções.

O músculo esquelético compõe-se de 40% do peso do corpo humano 1. Distúrbios associados com musculares representam uma imensa de saúde e dos custos económicos 2. Como este tecido altamente complexo e organizado é formado, mantidos e regenerado constitui um campo de pesquisa extensa e bem estabelecida. Dependendo do interesse científico específico, a abordagem mais adequada pode variar entre culturas miotubos simples aos mais complexos modelos in vivo 3 - 6. O objetivo deste protocolo é o de proporcionar um sistema in vitro que permite a monitorização de miogênese através de imagens ao vivo e imunofluorescência. Em comparação com as abordagens tradicionais, este sistema oferece uma visão muito completa e dinâmica no processo miogênica mouse. As células podem ser seguidas a partir da fase de mioblastos ao myofiber madura, multinucleadas exibindo tríades transversais e núcleos periféricos 7. Este nível de maturação podeser obtida utilizando equipamento de cultura celular normal, sem a necessidade de aparelhos estimuladores ou mecânicos complexos. Embora algumas bem sucedidas em sistemas in vitro têm sido relatados 8,9, a nosso conhecimento, este é o único protocolo de gerar fibras musculares maduras rato com a T-túbulos transversalmente emparelhado com Sarcoplasmic Reticulum (SR). Assim, este sistema in vitro pode ser utilizada para estudar os mecanismos moleculares da formação tríade, que são ainda pouco compreendidos 10. Uma outra vantagem da utilização deste sistema é a disponibilidade de recursos destinados-rato validadas, tais como anticorpos, drogas e instrumentos de ARNi. O protocolo relativamente simples não requer etapas trabalhosas, manipulação altamente qualificados, ou equipamentos caros e dedicado. Myofibers amadureceu começam a aparecer após 5 d de diferenciação de cultura 7, exibindo contratilidade juntamente com faíscas de cálcio (dados não publicados). Em uma semana, o desenvolvimento diferenteal fases de uma das células mais complexas no corpo de um mamífero pode ser estudada em combinação com uma variedade de ensaios in vitro.

<table><tbody><tr><td>Dispase II</td><td>Gibco</td><td>17105041</td><td></td></tr><tr><td>Collagenase&amp;nbsp;type V</td><td>Sigma-Aldrich-Aldrich</td><td>C9263</td><td></td></tr><tr><td>IMDM, Glutamax supplemented</td><td>Gibco</td><td>31980022</td><td></td></tr><tr><td>Matrigel Growth reduced factor</td><td>Corning</td><td>354230</td><td>protein concentration of the lot should be around 10 mg/mL and endotoxin result should be &amp;lt;1.5</td></tr><tr><td>Chicken Embryo Extract</td><td>MP biomedical</td><td>2850145</td><td>it is also possible to prepare in the lab (Danoviz ME, Yablonka-Reuveni Z. Methods Mol Biol (2012))</td></tr><tr><td>Recombinant rat agrin</td><td>R&amp;amp;D systems</td><td>550-AG-100</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Horse Serum</td><td>GE Healthcare&amp;nbsp;</td><td>11581831</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen</td><td>13778-150</td><td>used to transfect siRNA</td></tr><tr><td>Lipofectamine LTX</td><td>Invitrogen</td><td>15338-100</td><td>used to transfect DNA</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668030</td><td>used to transfect siRNA plus DNA</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190094&amp;nbsp;</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Eurobio</td><td>CVFSVF0001</td><td></td></tr><tr><td>Cell strainer</td><td>Corning</td><td>21008-949</td><td></td></tr><tr><td>Fluorodishes</td><td>World precision instruments</td><td>FD35-100</td><td>dishes used to cultivate cells for live imaging</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>16% PFA (Paraformaldehyde)</td><td>Science Services GmbH</td><td>E15710</td><td></td></tr><tr><td>Goat Serum</td><td>Sigma-Aldrich</td><td>G9023</td><td></td></tr><tr><td>BSA (Bovine Serum Albumine)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Saponine</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>use at 200 ng/&amp;micro;L</td></tr><tr><td>Fluoromount-G</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions and Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Digestion Mix</td><td></td><td></td><td>in DPBS&lt;br/&gt; 5 mg/mL collagenase&lt;br/&gt; 3.5 mg/mL dispase&lt;br/&gt; sterile filtered, can be stored in working aliquotes for 2 weeks at -20 &amp;deg;C</td></tr><tr><td>Dissection Medium</td><td></td><td></td><td>in IMDM Glutamax supplemented&lt;br/&gt; 10% FBS&lt;br/&gt; 1% Penicillin/Streptomycin&lt;br/&gt; sterile filtered</td></tr><tr><td>Growth Medium</td><td></td><td></td><td>in IMDM Glutamax supplemented&lt;br/&gt; 20% FBS&lt;br/&gt; 1% Chicken Embryo Extract&lt;br/&gt; 1% Penicillin-Streptomycin&lt;br/&gt; sterile filtered</td></tr><tr><td>Differentiation Medium</td><td></td><td></td><td>in IMDM Glutamax supplemented&lt;br/&gt; 2% Horse Serum&lt;br/&gt; 1% Penicillin-Streptomycin&lt;br/&gt; sterile filtered</td></tr><tr><td>Blocking Solution</td><td></td><td></td><td>in DPBS&lt;br/&gt; 10% Goat Serum&lt;br/&gt; 5% BSA&lt;br/&gt; add 0.1% saponine when incubating with primary and secondary antibodies</td></tr></tbody></table>

in vitro differentiation of mature myofibers for live imaging

Os músculos esqueléticos são compostos de miofibras, as maiores células no corpo de um mamífero e um dos poucos sincicios. Como as estruturas complexas e evolutivamente conservadas que o compõem são montados permanece sob investigação. Suas características de tamanho e fisiológicas, muitas vezes restringir aplicativos de manipulação e de imagem. A cultura das linhas celulares imortalizadas é amplamente usada, mas ela só pode replicar os primeiros passos de diferenciação. Aqui, descrevemos um protocolo que permite a manipulação genética fácil de miofibras provenientes de mioblastos principal do mouse. Após uma semana de diferenciação, as miofibras exibir contractilidade, alinhados sarcómeros e tríades, bem como núcleos periféricos. Todo o processo de diferenciação pode ser seguido por meio de imagens ao vivo ou imunofluorescência. Este sistema combina as vantagens de a existente ex vivo e em protocolos in vitro. A possibilidade de fácil e eficiente, assim como a transfecçãoa facilidade de acesso a todos os estágios de diferenciação amplia as aplicações potenciais. Miofibras podem subsequentemente ser utilizados não só para responder a questões de desenvolvimento e biologia celular relevantes, mas também para reproduzir fenótipos da doença muscular para aplicações clínicas.

A extensão do desenvolvimento da fibra muscular é em grande parte determinado pela pureza e viabilidade dos mioblastos isolados. A adesão, proliferação, e a capacidade de fusão pode ser utilizado para aceder empiricamente os parâmetros (Figura 1 A, B). No proliferação D2, mioblastos deveria ter respeitado e deve apresentar a forma típica fusiforme. Proliferação se espera que aconteça extensivamente nesta fase, que conduz à formação de miotubos espontânea no dia seguinte (Figura 1B). confluência celular pode precisar de pequenos ajustes. Deve ser aumentada se mioblastos tomar mais do que 3 d para proliferar e se fundem. Deve ser diminuída se miofibras não são deixadas a crescer e alongada relativamente simples devido à sua densidade. Confluência tipicamente diminui a partir do centro para a periferia do prato, de modo a melhor miofibras devem ser encontrados para as regiões exteriores. Miotubos rapidamente alongado e vários monitores núcleos alinhadas centralmente (Figura 1C). Por D5, algumas células começam a adquirir estrias e movendo-se os seus núcleos para a periferia. O número de miofibras maduras com características irá aumentar com o tempo, bem como com a espessura das células (Figura 1D). O grau de diferenciação pode ser mais observada por imunofluorescência. Miofibras fixos na diferenciação D8 tríades presentes transversais. Isto pode ser confirmado por componentes de imagem dos túbulos T (DPHR) e o SR (triadin), que se prevê venham a colocalize em tríades (Figura 2). A funcionalidade de miofibras podem ser tratadas por meio de imagens ao vivo. De diferenciação D3 em diante, as células apresentam espasmos espontâneos. Por transfecção de um sensor de cálcio (por exemplo,., GCaMP6f 11), é possível observar que as contracções são acoplados com picos de cálcio (Figura 3). Usando este sistema, fomos capazes de identificar um caminho molecular romance que é interrompido nas miopatias centronuclear e distrofias miotônica, que pode, portanto, ser um novo alvo para terapias moleculares inovadoras 7. Também se adaptaram este método para estudar o desenvolvimento da junção neuromuscular (JNM) 12. Através da co-cultura com ratos explantes da medula espinhal, descrevemos um papel para dineína no MNJ formação 13. <img alt="figura 1" src="/files/ftp_upload/55141/55141fig1.jpg" /> Figura 1: estágios de desenvolvimento da cultura mioblasto. A) A proliferação D2, mioblastos aderiram e começaram a proliferar. B) No prolifração D3, uma confluência de 60-80% é alcançado, e mioblastos começar a fundir espontaneamente. C) Em D3 diferenciação, miotubos contendo núcleos localizados centralmente são predominantes. D) A partir de diferenciação D5 em diante (por exemplo, dia 8), myofibers começar exibindo estrias e núcleos periféricos e começar a engrossar. Barra de escala: 50 ^ m. <a href="http://ecsource.jove.com/files/ftp_upload/55141/55141fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/55141/55141fig2.jpg" /> Figura 2: Representante confocal Imagem de um D8 miofibras imunocoloração. A) A imunocoloração para o receptor dihidropiridina (DHPR, painel superior) e triadin (TRDN, painel do meio). Uma sobreposição dos canais DHPR, TRDN e DAPI mostra co-localização dos componentes da tríade. B)<html> Um perfil de intensidade da linha amarela desenhado em A. C) Uma imagem 3D de renderização de volume de miofibras coradas para α-actinina (verde) e DAPI (azul). Escala de barra e grade width: 5 mm. <a href="http://ecsource.jove.com/files/ftp_upload/55141/55141fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/55141/55141fig3.jpg" /> Figura 3: Imagens ao vivo dos níveis de cálcio em miofibras com Spontaneous Twitching. A) de alta velocidade de lapso de tempo (20 ms quadros) microscopia de uma faísca de cálcio em um myofiber espasmos. Cálcio foi detectado através da expressão de GCaMP6f (Addgene plasmídeo # 40755). B) Quantificação da intensidade de fluorescência ao longo do tempo para o sensor de cálcio no painel A._blank &quot;&gt; Por favor clique aqui para ver uma versão maior desta figura.

Watch this Scientific Journal Video about In Vitro Differentiation of Mature Myofibers for Live Imaging at JoVE.com

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

Na diferenciação in vitro de miofibras maduras for Imaging ao vivo

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

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Research

JoVE Journal

Developmental Biology

In Vitro Differentiation of Mature Myofibers for Live Imaging

10.2K visualizações.  Universidade de Lisboa. Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

Vídeo: In Vitro Differentiation of Mature Myofibers for Live Imaging

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 of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

Isolamento de mioblastos humanos, avaliação da diferenciação miogênica e medição de entrada de cálcio armazenada

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Reprogramação Direta de Fibroblastos Humanos em Myoblasts para investigar terapias para distúrbios neuromusculares

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Genética

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Rastreamento de Dinâmica de enxerto muscular em animais de pequeno In Vivo Fluorescent Imagem

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Biologia

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolamento e Imortalidade de linhas celulares derivadas do paciente a partir de biópsia muscular para a doença Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Medicina

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 ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA Eletroporação, Isolamento e imagem das miofibras

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

Explorando a imagem ao vivo aos núcleos de pista durante a diferenciação de Myoblast e fusão

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Ensaio de cultura de myofibra única para a avaliação da funcionalidade de células-tronco musculares adultas Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

Medidas contráteis de alto rendimento de fibras musculares intactas de camundongos embutidas em hidrogel usando um sistema baseado em óptica

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...