Questo lavoro è stato parzialmente sostenuto da una sovvenzione di Fund-A-Fellow della Fondazione Myotonic Dystrophy (MDF-FF-2014-0013) e dello Scienziato per la Sviluppo dell&#39;American Heart Association (16SDG29320001).

I topi transgenici Wildtype e Nestin-GFP sono stati alloggiati nella struttura animale presso l&#39;Università del Minnesota. Tutte le procedure sperimentali sono state approvate dal Comitato istituzionale per la cura e l&#39;uso degli animali presso l&#39;Università del Minnesota e sono stati conformi alla Guida NIH per la cura e l&#39;uso degli animali da laboratorio. 1. Dissezione del muscolo e isolamento a cellule singole <ol><li> Eutanizzare i topi adulti (6-10 settimane, sia maschi che femmine) con il tribromoetanolo (250 mg / kg, ip) e sterilizzare la loro pelle dell&#39;addome con il 70% di etanolo. NOTA: il Tribromoetanolo è stato usato qui invece che la ketamina per l&#39;anestesia / eutanasia, in quanto la chetamina è nota per interagire con i recettori NMDA, che potrebbero avere un impatto sullo studio. </li><li> Posizionare i topi in posizione supina e utilizzare un bisturi per eseguire un&#39;incisione orizzontale sulla pelle addominale. Scorri la pelle manualmente, tirando in direzioni opposte per esporre i muscoli posteriori. </li><li> RaccolteI muscoli di entrambi i muscoli posteriori usando pinze e forbici. Conservarli in salina sterilizzata tamponata fosfato (PBS) integrata con 1% di penicillina-streptomicina (P / S) sul ghiaccio. </li><li> Lavare i muscoli dell&#39;indietro dissected in PBS ghiacciato-freddo completato con 1% P / S due volte e trasferirli in una piastra sterile da 10 cm. </li><li> Separare con cautela i nervi, i vasi sanguigni e il tessuto connettivo dai muscoli usando le pinze e le forbici sotto un microscopio di dissezione ad ingrandimento 2X. </li><li> Tagliare finemente e tagliare i muscoli in piccoli pezzi (1-2 mm 3 ) usando le forbici sterili e le lame. Se necessario, aggiungere una piccola quantità di Dulbecco&#39;s Modified Eagle Medium (DMEM) per assicurare che i muscoli non venissero asciugati. </li><li> Determinare meccanicamente il tessuto pipettando su e giù attraverso una pipetta serologica da 10 ml 10 volte. </li><li> Aggiungere la soluzione di digestione appena fatta (DMEM integrata con 0,2% di collagene tipo 2) alla miscela. Incubare a 37 ° C per 2 ore con gentlAgitazione a 35 giri / min. </li><li> Tritare usando un ago da 18 G per omogeneizzare la miscela. Quindi, centrifugare a 500 xg per 5 min. Scartare il surnatante e quindi risospendere il pellet nello 0,25% di tripto / EDTA. Incubare a 37 ° C per 10 min. Ripetere la fase di centrifugazione ancora due volte. </li><li> Aggiungere 10 ml di DMEM completato con 20% di siero fetale fetale (FBS) alla soluzione e centrifugare a 500 xg per 5 min. Scartare il surnatante e risospendere il pellet nel tampone di lisi di globuli rossi (155 mM NH 4 Cl, 10 mM KHCO3 e 0,1 mM EDTA). </li><li> Centrifugare a 500 xg per 5 min. Scartare il surnatante e risospendere il pellet nel tampone di smistamento (20 mM HEPES, pH 7,0, 1 mM EDTA e 1% BSA in PBS Ca / Mg 2+ , pH 7,0). Filtrare la miscela attraverso un filtro da 40 μm per ottenere una sospensione a singola cellula. </li><li> Centrifugare a 500 xg per 5 min. Scartare il supernatante e risospendere il pellet in 1 mL di tampone di smistamento. </li><li> Conti ilNumero di cellule con un emocitometro e diluire la sospensione a singola cellula a 5 x 10 6 / mL nel tampone di smistamento. </li></ol> 2. Colorazione e ordinamento delle cellule <ol><li> Preparare i controlli e il campione come descritto nella tabella 1 . Macellare la sospensione a singola cellula con i rispettivi anticorpi sul ghiaccio per 30 minuti, come descritto nella tabella 1 . </li><li> Centrifugare a 500 xg per 5 minuti e lavare i pellet due volte con il tampone di smistamento. </li><li> Aggiungere DAPI alle soluzioni a cellule singole, come indicato nella tabella 1. Utilizzare 5 μg / mL DAPI (concentrazione finale) per il controllo monocromatico DAPI e 1 μg / mL DAPI per il controllo PDGFRβ-PE-FMO e il campione. Tenere tutti i tubi sul ghiaccio durante l&#39;esperimento. </li><li> Accendere il destinatario e il software. Eseguire la scansione e inserire un chip di classificazione da 100 μm quando richiesto. </li><li> Eseguire l&#39;impostazione automatica ( ad esempio l&#39; allineamento del chip, la calibrazione della gocciolina, la calibrazione dei flussi laterali e il ritardo di ordinamento calibratiOn) caricando le perline di impostazione automatica quando richiesto. </li><li> Una volta completata l&#39;installazione automatica, vai alla scheda &quot;Esperimento&quot;, fai clic su &quot;Nuovo&quot; e seleziona &quot;Modello vuoto&quot; da &quot;Modelli pubblici&quot;. </li><li> Sotto &quot;Impostazioni di misura&quot; immettere &quot;DAPI&quot; per &quot;FL1&quot;, &quot;Nestin-GFP&quot; per &quot;FL2&quot; e &quot;PDGFRβ&quot; per &quot;FL3.&quot; Deselezionare le caselle per &quot;FL4&quot; - &quot;FL6&quot;. </li><li> Controlla le caselle per attivare i laser 405, 488 e 561 e fai clic su &quot;Crea nuovo esperimento&quot;. </li><li> Selezionare l&#39;opzione &quot;Start Compensation Wizard&quot; e seguire le istruzioni software &quot;Compensation Wizard&quot; per impostare la compensazione. <ol><li> Carica il controllo non conservato e fai clic su &quot;Avvia&quot;. Fare clic su &quot;Detector &amp; Threshold Settings&quot; e regolare il guadagno del sensore dei sensori FSC e BSC per mettere la popolazione sulla bilancia. </li><li> Anno DominiSolo i livelli di guadagno dei flussi di fluorescenza FL1-FL3 per posizionare le popolazioni negative sul lato sinistro degli istogrammi. Fare clic sul pulsante &quot;Registra&quot; per registrare i dati. </li><li> Caricare controlli monocromatici uno a uno quando richiesto. Fai clic su &quot;Avviare e registrare&quot; per registrare i dati. Regolare i cancelli per le popolazioni positive sugli istogrammi. Fai clic su &quot;Avanti&quot;. </li><li> Vai a &quot;Calcola Matrice&quot; nella scheda &quot;Compensi&quot; e fai clic su &quot;Calcola&quot; nel pannello &quot;Calcola impostazioni di compensazione&quot; per eseguire la compensazione. Fai clic su &quot;Fine&quot; per uscire dalla &quot;Creazione guidata remunerazione&quot;. </li></ol></li><li> Caricare il controllo PDGFRβ-PE-FMO e fare clic su &quot;Start&quot;. Disegna una porta poligonale (Porta A) attorno alle celle di interesse sotto il grafico &quot;Tutti gli eventi&quot;. </li><li> Fare doppio clic all&#39;interno della porta A per creare una trama figlia. Cambiare l&#39;asse Y su DAPI e disegnare una porta poligonale (Porta B) attorno alle celle viventi (DAPI low ). Fare doppio clic su iNside Gate B per creare una trama figlia. Cambiare l&#39;asse X su FSC-H e l&#39;asse Y a FSC-W e disegnare una porta poligonale (Porta C) attorno alle singole per eliminare i dublettini. </li><li> Fare doppio clic all&#39;interno della porta C per creare una trama figlia. Cambiare l&#39;asse X a Nestin-GFP e l&#39;asse Y a PDGFRβ-PE. Fare clic sul pulsante &quot;Registra&quot; per registrare i dati. </li><li> Caricare il campione e ripetere i passaggi 2.11-2.12. Dopo la registrazione, fai clic su &quot;Pausa&quot; per conservare il campione. </li><li> Definire i limiti di gating per le cellule PDGFRβ + e Nestin-GFP + basate sul controllo PDGFRβ-PE-FMO. Disegnare le porte per le popolazioni di PDGFRβ + Nestin-GFP e PDGFRβ + Nestin-GFP + . </li><li> Sotto &quot;Metodo di ordinamento&quot;, selezionare &quot;Tubi a 2 vie&quot; e assegnare le celle &quot;PDGFRβ + Nestin-GFP&quot; e &quot;PDGFRβ + Nestin-GFP + &quot; ai tubi di raccolta destro e sinistrospettivamente. Montare i tubi di raccolta 15 ml di riempimento con tampone di riempimento nella fase di raccolta e fare clic sul pulsante &quot;Load Collection&quot;. </li><li> Fai clic sul pulsante &quot;Riprendi&quot; per mantenere il campione in esecuzione. Fare clic su &quot;Start Sort&quot; per raccogliere le cellule PDGFRβ + Nestin-GFP (periciti di tipo I) e PDGFRβ + Nestin-GFP + (periciti di tipo II). </li></ol> 3. Analisi post-ordinamento <ol><li> Centrifugare le cellule ordinate a 500 xg per 5 minuti, risospendere il pellet in 1 mL di mezzo di pericce (vedere tabella dei materiali ) e contare la densità cellulare usando un emocitometro. </li><li> Periciti di tipo I e II di tipo su copertine rivestite con poli-D-lisina (PDL) a ~ 1 x 10 4 cellule / cm 2 . Crescere in media pericta per 3 giorni a 37 ° C con 5% CO 2 . </li><li> Il giorno 3, esaminare la morfologia pericita (sotto il contrasto di fase) e l&#39;espressione endogena di Nestin-GFP utilizzando un micro fluorescenteCampo di applicazione (laser di eccitazione: 488 nm, filtro di eccitazione: 470/40 nm e filtro di emissione: 515/30 nm). Prendere le immagini sotto un obiettivo 20X (0,45 NA). </li><li> Sostituire il mezzo di pericite con adipogenic (mezzo di base basale MSC mouse + supplemento stimolatorio adipogenico) e myogenic (DMEM + 2% cavallo di cavallo) per iniziare rispettivamente la differenziazione adipogenica e miogenica, come descritto in precedenza 20 . Cambiare il supporto ogni 2-3 giorni. </li><li> Fissare le cellule il giorno 17 (14 giorni dopo la differenziazione adipogenica / miogenica) in 4% di paraformaldeide (PFA) per 20 minuti a temperatura ambiente. NOTA: Attenzione, PFA è un cancerogeno. </li><li> Eseguire l&#39;immunocitochimica contro la perilipina (marcatore di adipociti) e la S-myosina (marcatore miootubo / miofibre maturo), come descritto nelle pubblicazioni precedenti 19 , 20 . <ol><li> Lavare le cellule fisse 3 volte in PBS per 10 minuti a temperatura ambiente. </li><li> Aggiungere buffer di blocco (PBSCompleta di siero di asina al 5%, 3% BSA e 0,3% Triton X-100) e incubare a temperatura ambiente per 1 ora. </li><li> Incubare le cellule con anticorpi anti-perilipina (2 μg / mL) e / o anti-S-myosina (2 μg / mL) a 4 ° C per una notte. </li><li> Lavare le cellule 3 volte in PBS per 10 minuti a temperatura ambiente. </li><li> Incubare le cellule con anticorpi anti-coniglio di Alexa 555 (4 μg / mL) e / o Alexa555 anti-mouse (4 μg / mL) a temperatura ambiente per 1 ora. </li><li> Lavare le cellule 3 volte in PBS per 10 minuti a temperatura ambiente. </li><li> Montare le cellule immunocoste con supporto di supporto contenente DAPI (vedere la tabella dei materiali). Esaminare la perilipina e l&#39;espressione di S-myosina usando un microscopio fluorescente (laser di eccitazione: 543 nm, eccitazione 540/45 nm e emissione di 600/50 nm) e fotografare con un obiettivo 40X (0,60 NA). </li></ol></li></ol>

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Periciti sono cellule perivascolari multipotenti 22 , 23 situate sulla superficie abluminale dei capillari 21 , 26 . Nei muscoli scheletrici, i periciti sono in grado di differenziarsi lungo i percorsi adipogenici e / o miogenici 19 , 20 , 24 . Recenti studi hanno rivelato due sottopopolazioni di periciti, con d...

La distrofia muscolare è un disturbo muscolare-degenerativo che non ha finora trattamenti efficaci. Lo sviluppo di terapie che promuovono la rigenerazione dei tessuti è sempre stato di grande interesse. La rigenerazione tissutale e la riparazione dopo danni dipendono da cellule staminali residui / cellule progenitrici 1 . Le cellule satellitari sono impegnate da cellule precursori myogeniche che contribuiscono alla rigenerazione muscolare 2 , 3 , 4 , 5 , 6 , 7 . Tuttavia, il loro utilizzo clinico è ostacolato dalla loro limitata migrazione e dal basso tasso di sopravvivenza dopo l&#39;iniezione, nonché dalla loro diminuita capacità di differenziazione dopo l&#39;amplificazione in vitro 8 , 9 , 10 , 11 . Oltre ai satellitiLe cellule ei muscoli scheletrici contengono anche molte altre popolazioni cellulari con potenzialità myogenica 12 , 13 , 14 , 15 , 16 come le cellule interstiziali posizionali di fattore di crescita del recettore beta-beta (PDGFRβ) derivato dalle piastrine. Ci sono prove che dimostrano che le cellule PDGFRβ + derivate dal muscolo sono in grado di differenziarsi in cellule miogeniche e migliorare la patologia muscolare e la funzione 14 , 17 , 18 , 19 , 20 . PDGFRβ prevalentemente etichetta periciti 21 , che sono cellule perivascolari con pluripotenza 22 , 23 . Oltre al PDGFRβ, molti altri marcatori, tra cui Neuron-Glial 2 (NG2) e CD146, sono utilizzati anche per iDentify pericytes 21 . Va comunque osservato che nessuno di questi marcatori è specifico per cicli 21 . Recenti studi hanno rivelato due sottotipi di periciti muscolari, chiamati tipo I e tipo II, che esprimono diversi marcatori molecolari e svolgono funzioni distinte 19 , 24 , 25 . Biochimicamente, le pericelle di tipo I sono NG2 + Nestin, mentre le pericelle di tipo II sono NG2 + Nestin + 19 , 24 . Funzionariamente, i periciti di tipo I possono subire differenziazione adipogenica, contribuendo all&#39;accumulo di grassi e / o alla fibrosi, mentre i periciti di tipo II possono differenziarsi lungo il percorso miocogenico, contribuendo alla rigenerazione muscolare 19 , 24 , 25 . Questi risultati dimostrano che: (1) i periciti di tipo I possono bE destinato al trattamento di disturbi degenerativi grassi / fibrosi, e (2) le pericie di tipo II hanno un grande potenziale terapeutico per la distrofia muscolare. Ulteriori indagini e caratterizzazione di queste popolazioni richiedono un protocollo di isolamento che consente la separazione dei periciti di tipo I e tipo II ad un alto livello di purezza. Attualmente, l&#39;isolamento delle sottopopolazioni pericite si basa sui topi doppi transgenici NG2-DsRed e Nestin-GFP 19 , 24 . La disponibilità di topi NG2-DsRed e la qualità della maggior parte degli anticorpi NG2 limitano l&#39;uso diffuso di questo metodo. Dato che tutti i periciti NG2 + esprimono PDGFRβ nei muscoli scheletrici 19 , 20 , 24 , ipotizziamo che NG2 può essere sostituito da PDGFRβ per l&#39;isolamento delle periciti e delle loro subpopulazioni. Questo lavoro descrive un protocollo basato su FACS cheUtilizza la colorazione PDGFRβ e il segnale Nestin-GFP. Questo metodo è meno impegnativo per gli investigatori perché: (1) non richiede lo sfondo NG2-DsRed e (2) utilizza gli anticorpi PDGFRβ commercialmente disponibili, ben caratterizzati. Inoltre, consente l&#39;isolamento simultaneo di periciti di tipo I e di tipo II ad alta purezza, permettendo di approfondire la biologia e il potenziale terapeutico di queste sottopopolazioni pericite. A seguito della purificazione, queste cellule possono essere coltivate in coltura e le loro morfologie possono essere visualizzate. Questo lavoro mostra anche che i periciti di tipo I e II tipo isolati utilizzando questo metodo, come quelli purificati da topi doppi transgenici, sono rispettivamente adipogenici e miogenici.

<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

Periciti sono cellule multipotenti perivascolari che mostrano eterogeneità in organi diversi o addirittura all&#39;interno dello stesso tessuto. Nei muscoli scheletrici esistono almeno due sottopopolazioni pericite (chiamate tipo I e tipo II) che esprimono differenti marcatori molecolari e hanno differenti capacità di differenziazione. Utilizzando i topi a doppio-transgenici NG2-DsRed e Nestin-GFP, le pericelle di tipo I (NG2-DsRed + Nestin-GFP-) e di tipo II (NG2-DsRed + Nestin-GFP + ) sono state isolate con successo. Tuttavia, la disponibilità di questi topi doppi transgenici impedisce l&#39;uso diffuso di questo metodo di purificazione. Questo lavoro descrive un protocollo alternativo che consente l&#39;isolamento facile e simultaneo di periciti tipo I e II di tipo muscolare scheletrico. Questo protocollo utilizza la tecnica di selezione delle cellule attivata con fluorescenza (FACS) e mira a PDGFRβ, piuttosto che a NG2, insieme al segnale Nestin-GFP. Dopo l&#39;isolamento, digitare I e tyPe periciti mostrano morfologie distinte. Inoltre, le pericie di tipo I e tipo II isolate con questo nuovo metodo, come quelle isolate dai topi doppi transgenici, sono rispettivamente adipogenici e miogenici. Questi risultati suggeriscono che questo protocollo può essere utilizzato per isolare le sottopopolazioni pericite dai muscoli scheletrici e, probabilmente, da altri tessuti.

I parametri FACS, compresa l&#39;intensità laser e la compensazione dei canali, vengono corretti in base ai risultati di controlli non controllati e controlli monocromatici. Il controllo PDGFRβ-PE-FMO viene utilizzato per impostare il gating per la popolazione di PDGFRβ-PE + ( Figura 1A ). Tra le cellule PDGFRβ-PE, due popolazioni che rappresentano le cellule Nestin-GFP + e Nestin-GFP sono chiaramente separate ( <strong class="xfig"...

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

Questo lavoro descrive un protocollo basato su FACS che consente l&#39;isolamento facile e simultaneo di periciti tipo I e II di tipo muscolare scheletrico.

Isolamento delle pericelle di tipo I e II di muscoli scheletrici del mouse

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

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

9.1K Views.  University of Minnesota.  Questo lavoro descrive un protocollo basato su FACS che consente l'isolamento facile e simultaneo di periciti tipo I e II di tipo muscolare scheletrico. 

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.

Isolamento e quantitativa immunocitochimica caratterizzazione di cellule miogeniche primarie e fibroblasti da muscolo scheletrico umano

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

Ricerca

Biologia dello sviluppo

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 differenziazione dei mioblasti primari dagli esopanti muscolari scheletrici del topo

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 di cellule endoteliali microvascolari murino primario del muscolo scheletrico

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

Neuroscienze

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 la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

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 trapianto di cellule muscolari satellite

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 di polmonare cellule muscolari lisce di topi neonati

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 purificazione dei Periciti Cardiaci Murine

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 della miofibra singola da un modello Murine della distrofia muscolare di Emery-Dreifuss nei primi sviluppi post-Natal

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 metodo ad alta uscita per isolare i periciti cerebrali dal mouse

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 di popolazioni di cellule staminali quiescenti da singoli muscoli scheletrici

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