1. Tessuto Collezione: <ol><li> Utilizzare topi tra 7 e 12 settimane di età. Di solito, almeno due animali sono utilizzati: uno è quello di impostare il FACS monocolore controlli necessari per il risarcimento e per campioni isotipo fluorescenza-meno-un controllo. L&#39;altro è il campione reale per l&#39;ordinamento. </li><li> Euthanize i topi da CO 2 inalazione o secondo un protocollo di etica della scelta. Topi posto su un tovagliolo di carta, rivolta verso l&#39;alto, e spruzzare con il 70% di etanolo a fondo per evitare la contaminazione con la pelliccia del mouse. </li><li> Sollevare la cute nella regione addominale con una pinza, poi fare un piccolo taglio longitudinale con forbici affilate. </li><li> Sbucciate i due lembi di pelle in direzioni opposte (su e giù) per esporre completamente i muscoli degli arti posteriori sotto. </li><li> Dell&#39;accisa il tessuto muscolare inserendo ed estendendo le forbici lungo entrambi i lati della tibia. </li><li> Ripetere la stessa procedura per il femore per estrarre il tessuto muscolare. </li><li> Mettere tutti i tessuti muscolari asportati in 60 millimetri di Petri-piatti, utilizzando un piatto per il tessuto di ogni topo. </li><li> Ripetere quanto sopra per tutti i mouse. </li></ol> 2. Dissociare tessuto in cellule singole per digestione enzimatica: In questa sezione del protocollo, utilizzare i reagenti e strumenti sterili e lavorare in condizioni sterili. <ol start="9"><li> Aggiungere 2 ml di Collagenasi II (Sigma, cat # 6885, 500U / mL) e 20 l di 250mm CaCl 2 magazzino ad ogni Petri-piatto. </li><li> Lacerare il tessuto muscolare in piccoli pezzi (circa 1 mm 3) con due pinze (una seghettato, una con 2 denti, da Strumenti Scienze Arti, cat # 11051-10, 11154-10), facendo attenzione a rimuovere ed eliminare il più contaminati non-tessuto muscolare possibile. </li><li> Coprire il Petri-piatti e incubare loro a 37 gradi centigradi per 30 minuti. </li><li> Recuperare il Petri-piatti e usare una siringa da 3 ml stantuffo a schiacciare a fondo i pezzi di tessuto. Utilizzare uno stantuffo per ogni campione per evitare contaminazioni. </li><li> Aggiungete un po &#39;(~ 5 ml) PBS freddo sterile ad ogni Petri-piatto e trasferire i fanghi tessuto in un tubo 50mL Falcon (sciacquare la Petri-piatto, se necessario, di recuperare tutti i tessuti) </li><li> Agitare la provetta Falcon vigorosamente, riempire la provetta con PBS, centrifugare @ 800 giri x 5 minuti (da banco Eppendorf top Modello: 5810R), e aspirare il surnatante (ripetere 3 volte) </li><li> Aggiungere 1 ml di miscela Collagenasi D (Roche, Cat # 11088882001, 1,5 U / mL) / dispasi II (Roche, Cat #: 04942078001, 2.4u / mL) e 10μL di CaCl 2 (250mm) a ciascuna provetta, incubare a 37 gradi Celsius con rotazione per un&#39;ora. </li><li> Aggiungere 5-10 ml di freddo, sterile PBS, vortice e pipetta accuratamente per omogeneizzare il tessuto. </li><li> Riempire i tubi con PBS e mescolare bene. </li><li> Trasferire il liquido in un colino cellula 40μm collocato su un tubo 50 ml Falcon per filtrare i pezzi di altri grandi produttori di tessuto. Dividere ogni campione uniformemente in tre 50mL tubi Falcon. Riempire ciascuno dei tubi con PBS quindi si centrifuga @ 1600rpm per 5 minuti a 8 gradi Celsius. </li><li> Eliminare il supernatante e raccogliere le cellule per la colorazione. </li></ol> 3. Anticorpi colorazione e ordinamento: In questa sezione del protocollo, l&#39;uso FACS tamponi sterili e la raccolta e il lavoro in condizioni sterili. Anticorpi commerciali sono generalmente considerati sterili se correttamente gestita. <ol start="20"><li> Per singolo colore e campioni di controllo isotipico, risospendere il campione in 1 ml di buffer FACS e dividere le cellule sospese in nove 1,5 ml di pre-etichettati tubi Eppendorf (100 l ogni tubo). <ol><li> Impostare colorazione singolo colore si mescola in base alla seguente tabella: ATTENZIONE: La concentrazione finale di anticorpi isotipo di controllo nella macchia dovrebbe corrispondere a quello dell&#39;anticorpo primario sono il controllo. Per esempio, se anti-Sca-1 è usato per 1 μg/10 6 celle, il suo isotipo corrispondente deve essere utilizzato alla stessa concentrazione. <table border="1"><tbody><tr><td> Tubo # </td><td> Nome del tubo </td><td> Azienda </td><td> Cat # </td><td> clone </td><td> isotipo </td><td> diluizione </td></tr><tr><td> 1 </td><td> Non macchia </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2 </td><td> Hoechst33342 </td><td> sigma </td><td> B2261 </td><td></td><td></td><td> 2-5ug/ml </td></tr><tr><td> 3 </td><td> PI </td><td> sigma </td><td> P4864 </td><td></td><td></td><td> 1ug/ml </td></tr><tr><td rowspan="2"> 4 </td><td rowspan="2"> CD31-FITC CD45-FITC </td><td> eBioscience </td><td> 11-0311-85 </td><td> 390 </td><td> Rat IgG2a </td><td> 500 </td></tr><tr><td> Fatti in casa </td><td></td><td> I3 / 2 </td><td> Rat IgG2b </td><td> 500 </td></tr><tr><td> 5 </td><td> SCA1-PECY7 </td><td> eBioscience </td><td> 25-5981-82 </td><td> D7 </td><td> Rat IgG2a </td><td> 5000 </td></tr><tr><td> 6 </td><td> α-7 APC </td><td> Fatti in casa </td><td></td><td> R2F2 </td><td> Rat IgG2b </td><td> 1000 </td></tr></tbody></table></li><li> Impostare colorazione controllo isotipico mescola con la seguente tabella: <table border="1"><tbody><tr><td> Tubo # </td><td> Nome di tubo </td><td> Hoechst </td><td> PI </td><td> CD31- FITC </td><td> CD45- FITC </td><td> Scal- PE-Cy7 </td><td> A-7- APC </td><td> Rat- IgG2b- APC </td><td> Rat- IgG2a- pecy7 </td><td> Rat- IgG2a- FITC </td><td> Rat- IgG2b- FITC </td></tr><tr><td> 7 </td><td> Iso- CD31 / CD45 </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td></tr><tr><td> 8 </td><td> Iso- Scal- pecy7 </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td></tr><tr><td> 9 </td><td> Iso- α-7-APC </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td><td> - </td></tr></tbody></table></li><li> Pipettare il singolo colore e di isotopi mescola colorazione di controllo (di solito regolare la concentrazione di anticorpi in modo che tra 5 e 10 ml di colorazione miscela vengono aggiunti ad ogni campione) nel tubo Eppendorf appropriato dal punto 20. Mescolare bene e incubare in ghiaccio per 1 ora. </li></ol></li><li> Macchia cellule di topo campione con 800μL del cocktail di anticorpi (in basso) per l&#39;ordinamento delle cellule FACS. Mescolare bene e incubare in ghiaccio per 1 ora. Anticorpi cocktail: Hoechst, PI, CD31-FITC, CD45-FITC, SCA1-PECY7, α-7 APC ATTENZIONE: Il rapporto ottimale di anticorpi contro le cellule devono essere determinata individualmente ogni titolazione degli anticorpi, in quanto possono esistere differenze significative tra i lotti. La quantità ottimale di anticorpi da utilizzare devono essere espressi in μgs di anticorpi per 10 6 cellule bersaglio e mantenuta costante durante l&#39;utilizzo dello stesso lotto di anticorpi. Chiaramente, se il numero di cellule bersaglio non cambia significativamente tra gli esperimenti, o se il volume colorazione è in scala con il numero di cellule, una diluizione fisso di ciascun anticorpo può essere utilizzato anche. </li><li> Lavaggio: Aggiungere circa 1 ml di buffer di FACS per ognuna delle 9 provette di controllo, aggiungere circa 20 mL di tampone FACS alla provetta. </li><li> Centrifugare le provette Eppendorf @ 3000rpm per 5 minuti (da banco centrifuga Eppendorf in alto, Modello: 5417C). Centrifugare la provetta @ 1600rpm per 5 minuti (da banco centrifuga Eppendorf in alto, Modello: 5810R). </li><li> Aspirare e scartare il supernatante da tutti i tubi. </li><li> Risospendere le cellule nel buffer FACS (1 ml per ciascuno dei controlli, da 4 ml per il campione ordinamento), le cellule Pipettare in &quot;tubi FACS&quot; attraverso il filtro delle cellule tappo (cat BD Falcon # 352235, 40μm dimensioni dei pori) per rimuovere macchie rimanenti che possono influire sul citometro. </li><li> Impostare il FACS sorter con controlli singolo colore e controlli isotipo. </li><li> Raccogliere le cellule ordinati secondo la tabella seguente in un buffer di 3,5 ml di raccolta (95% DMEM, 5% FBS) separatamente. Normalmente, quando la raccolta entrambi gli arti posteriori, un mouse di tipo selvatico potrebbe produrre circa 150.000 cellule progenitrici miogeniche o 100.000 cellule progenitrici adipogenico, con la vitalità superiore al 95%, come giudicato da rianalizzati cellule ordinati per FACS al termine della procedura (Figura 1 ) Definizione dei progenitori miogenici e adipogenico base alla colorazione della superficie <table border="1"><tbody><tr><td> Anticorpo / macchia </td><td> Progenitrici miogenici (MP) </td><td> Progenitrici adipogenico (AP) </td></tr><tr><td> Hoechst </td><td> + </td><td> + </td></tr><tr><td> PI </td><td> - </td><td> - </td></tr><tr><td> CD31-FITC </td><td> - </td><td> - </td></tr><tr><td> CD45-FITC </td><td&gt; - </td><td> - </td></tr><tr><td> SCA1-PECY7 </td><td> - </td><td> + </td></tr><tr><td> α-7 APC </td><td> + </td><td> - </td></tr></tbody></table></li></ol> 4. Trapianto: <ol start="28"><li> Centrifugare le cellule ordinati @ 1500rpm per 5 minuti, togliere le cellule surnatante e trasferimento in autoclave i tubi Eppendorf (1,5 ml), lavare le cellule con 500 l di PBS sterile (i livelli di free!), Quindi si centrifuga @ 3000rpm per 5 minuti. Il mantenimento di tutti i reagenti in condizioni sterili e lavorare in un cappuccio colture di tessuti, risospendere progenitori miogenici in PBS, o progenitrici adipogenico in Matrigel (BD cat # 354234) a circa 10 6 cellule / ml. </li><li> Trapianto di progenitori miogenici o progenitrici adipogenico ai topi destinatario. <ol><li> Anestetizzare il mouse con isoflouran secondo la vostra politica istituzione. </li><li> Radersi i capelli intorno alla regione di iniezione, poi sterilizzare la pelle strofinando con etanolo al 70% </li><li> Iniettare 20μL dei progenitori miogenici o progenitori adipogenico (= 20k cellule) con un 3 / 10 siringa da insulina CC (cat BD # 309300). </li></ol></li><li> Dopo un tempo adeguato (tre settimane funziona bene, ma miofibre transgenici che esprimono i marcatori del donatore-derivati ​​sono di solito evidenti entro una settimana dopo il trapianto), anestetizzare i topi destinatario da passaggio 29 mediante iniezione intraperitoneale di 400 microlitri Avertin (Cat Sigma # T04840-2, 25 mg / ml), poi profumato transcardially con 50mL PBS (contiene 10 mM di EDTA) per primo, seguito da 15 ml del 4% PFA. Raccogliere tessuto bersaglio, post-fix con 4% PFA durante la notte, se necessario, poi il trasferimento al 20% di saccarosio durante la notte. Incorpora in ottobre, congelare e cryosection. </li></ol> 5. Rappresentante dei risultati: MP quando vengono trapiantate nel muscolo scheletrico del mouse, che prontamente si fondono con pre-esistente miofibre. Pertanto, qualsiasi etichetta genetico presente nelle cellule del donatore sarà facilmente individuabile nelle fibre che li ha accolti. La figura 2 mostra un esempio in cui le cellule trapiantate espresso umane phostphatase alcalino, ha rivelato istochimicamente. AP quando vengono trapiantate il risultato dipende in larga misura l&#39;ambiente del sito trapianto: quando queste cellule trapiantate per via sottocutanea dare origini a adipociti e miofibroblasti (vedi figura 2). In molti altri siti, compresi i muscoli, non sopravvivono a meno degenerazione grassa è stata indotta 3. <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="Figura 1" /> Figura 1. Ordinamento strategia per l&#39;isolamento delle popolazioni progenitrici dal muscolo scheletrico (A) strategia Ordinamento:. Cellule vitali sono state identificate sulla base di dispersione in avanti e side scatter. Colorazione Hoechst è stato utilizzato per escludere detriti anuclear e ioduro di propidio (PI) colorazione di escludere le cellule morte. Ematopoietiche (CD45) ed endoteliali (CD31), le cellule sono state escluse dalle porte di smistamento. Il α7 + Hoechst + PI - CD45 - CD31 - Scal - sottoinsieme contiene tutti i progenitori miogenici (MP). Il Scal + Hoechst + PI - CD45 - CD31 - α7 - popolazione contiene progenitori adipogenico (AP). (B) Fluorescenza-minus-one (FMO) controlla isotipo confermare la specificità della macchia. (C) Purezza controlli di sottoinsiemi MP e AP dopo l&#39;ordinamento. <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="Figura 2" /> Figura 2. Risultati rappresentativi seguenti MP e AP di trapianto. AP sono stati trapiantati per via sottocutanea (pannello superiore) e MP per via intramuscolare (pannello inferiore). In entrambi i casi, le cellule del donatore origine da un topo transgenico che esprime fosfatasi alcalina umano, identificato dalla colorazione marrone.

<ol>
	<li>Buckingham, M., Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+stem+cells.">Skeletal muscle stem cells.</a> Curr Opin Genet Dev. 18, 330-336 (2008).</li><li>Relaix, F., Marcelle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> Curr Opin Cell Biol. 21, 748-753 (2009).</li><li>Joe, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nat Cell Biol. 12, 153-163 (2010).</li><li>Natarajan, A., Lemos, D. R., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors:+A+double-edged+sword+in+skeletal+muscle+regeneration.">Fibro/adipogenic progenitors: A double-edged sword in skeletal muscle regeneration.</a> Cell Cycle. 9, (2010).</li><li>Sherwood, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adult+mouse+myogenic+progenitors:+functional+heterogeneity+of+cells+within+and+engrafting+skeletal+muscle.">Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.</a> Cell. 119, 543-554 (2004).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. , (2008).</li><li>Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

L&#39;obiettivo di questo protocollo è quello di trovare un equilibrio ragionevole tra alti rendimenti e alti vitalità delle cellule purificate. La fase più critica per garantire che le cellule sane sono state recuperate è, prevedibilmente, la dissociazione enzimatica del tessuto di partenza. Il trattamento del tessuto deve essere particolarmente delicato, che è difficile da dimostrare o apprezzare anche in un formato audiovisivo. Un altro fattore chiave è la durata della procedura. Più a lungo ci vuole per andare dal donatore al ricevente l&#39;animale, minore è la vitalità e, quindi, l&#39;efficienza attecchimento. In caso di domanda sulla vitalità che non è immediatamente risposto, cercando alla frequenza di eventi PI positivi nei campioni di cellule purificate dopo l&#39;ordinamento, si consiglia un test di diluizione limita a misurare clonogenicità viene eseguita 3. Sorta tipici muscolo danneggiato regolarmente resa di circa 1 in 15-20 cellule in grado di avviare colonie miogenici o fibro / adipogenico in vitro. Mentre praticamente del 100% delle colonie avviato da parlamentari contenere miotubi differenziati, solo circa un terzo delle colonie ottenute da FAP contenere adipociti oltre a miofibroblasti actina del muscolo liscio positivi.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Collagenasi II </td><td> Sigma-Aldrich </td><td> C-6885, </td><td> 500 U / ml </td></tr><tr><td> Collagenasi D </td><td> Roche </td><td> 11088882001 </td><td> 1,5 U / ml </td></tr><tr><td> Spostare II </td><td> Roche </td><td> 04942078001 </td><td> 2,4 u / ml </td></tr><tr><td> cellula filtro </td><td> BD </td><td> 352340 </td><td> 40 micron </td></tr><tr><td> Tubo con filtro cellulare </td><td> BD </td><td> 352235 </td><td> 40 micron </td></tr><tr><td> Hoechst33342 </td><td> Sigma-Aldrich </td><td> B2261 </td><td> 2-5 ug / ml </td></tr><tr><td> CD31-FITC </td><td> Ebiosciences </td><td> 11-0311-85 </td><td> Clone 390 </td></tr><tr><td> CD45-FITC </td><td> Fatto in casa </td><td></td><td> Clone I3 / 2 </td></tr><tr><td> SCA1-PECY7 </td><td> Ebiosciences </td><td> 25-5981-82 </td><td> Clone D7 </td></tr><tr><td> α-7 integrina APC </td><td> Fatto in casa </td><td> Informarsi con Rossi laboratorio </td><td> R2F2 </td></tr></tbody></table>

purification of progenitors from skeletal muscle

Muscolo scheletrico contiene popolazioni progenitore più di distinte origini embrionale e potenziale di sviluppo. Progenitori miogenici, di solito risiede in una &quot;posizione delle cellule satellite&quot; tra la membrana plasmatica miofibre e la laminina-ricchi membrana basale che ensheaths esso, sono auto-rinnovamento delle cellule che sono esclusivamente impegnati nel 1,2 lignaggio miogenico. Recentemente abbiamo descritto una seconda classe di progenitori nave associate in grado di generare e miofibroblasti adipociti bianchi, che risponde ai danni da entrare in modo efficace la proliferazione e fornisce supporto trofico per le cellule miogenici durante la rigenerazione dei tessuti 3,4. Uno dei test più affidabile per determinare il potenziale di sviluppo e rigenerazione di una popolazione di cellule dato si basa sul loro peso e il trapianto di 5-7. A tal fine abbiamo ottimizzato i protocolli per la loro purificazione mediante citometria di flusso dal muscolo dissociato enzimaticamente, che illustrerà in questo articolo. Le popolazioni ottenuti usando questo metodo contenere cellule colonia miogeniche o fibro / adipogenico formando: non altri tipi di cellule sono in grado di espandere in vitro o sopravvivere in consegna vivo. Tuttavia, quando queste popolazioni vengono utilizzati subito dopo l&#39;ordinamento per l&#39;analisi molecolare (ad es qRT-PCR) si deve tenere presente che i sottoinsiemi appena ordinati può contenere altre cellule agente inquinante che non hanno la capacità di formare colonie o engrafting destinatari.

Watch this Scientific Journal Video about Purification of Progenitors from Skeletal Muscle at JoVE.com

Purification of Progenitors from Skeletal Muscle

Metodo per la dissociazione enzimatica, l&#39;etichettatura e la purificazione della superficie mediante citometria a flusso di fibro / adipogenico e progenitori miogenici da topo muscolo scheletrico.

Purificazione di progenitori dal muscolo scheletrico

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

purification-of-progenitors-from-skeletal-muscle

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.2K Views.  University of British Columbia. Metodo per la dissociazione enzimatica, l'etichettatura e la purificazione della superficie mediante citometria a flusso di fibro / adipogenico e progenitori miogenici da topo muscolo scheletrico.

Video: Purification of Progenitors from Skeletal Muscle

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

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Preparazione e la cultura di cellule miogeniche Precursore / mioblasti primari dal muscolo scheletrico di adulti e esseri umani invecchiati

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

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 dei mioblasti umani, valutazione della differenziazione myogenica e misurazione dell&#39;entrata del calcio a livello di deposito

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

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

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

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

Isolamento, cultura, caratterizzazione e differenziazione delle cellule del progenitore muscolare umano dalla procedura di biopsia muscolare scheletrica

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

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

Isolare cellule staminali da tessuti molli muscoloscheletrici

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Biologia

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

FACS-isolamento e coltura di progenitori fibro-adipogeni e cellule staminali muscolari da muscolo scheletrico di topo imperturbato e ferito

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identificazione e isolamento di Oligopotent e dei progenitori mieloidi Lineage-commesso dal midollo osseo del topo

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Immunologia e infezioni

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 Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolamento dei vasi sanguigni derivati ​​da Multipotent precursori del muscolo scheletrico umano

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generazione di Myospheres Da hESCs di epigenetica Riprogrammazione

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Purificazione di progenitori dal muscolo scheletrico

Riepilogo

Esplora Altri Video

Capitoli in questo video

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

Preparazione e la cultura di cellule miogeniche Precursore / mioblasti primari dal muscolo scheletrico di adulti e esseri umani invecchiati

Isolamento dei mioblasti umani, valutazione della differenziazione myogenica e misurazione dell'entrata del calcio a livello di deposito

Isolamento, cultura, caratterizzazione e differenziazione delle cellule del progenitore muscolare umano dalla procedura di biopsia muscolare scheletrica

Isolare cellule staminali da tessuti molli muscoloscheletrici

FACS-isolamento e coltura di progenitori fibro-adipogeni e cellule staminali muscolari da muscolo scheletrico di topo imperturbato e ferito

Identificazione e isolamento di Oligopotent e dei progenitori mieloidi Lineage-commesso dal midollo osseo del topo

Isolamento, Cultura e trapianto di cellule muscolari satellite

Isolamento dei vasi sanguigni derivati da Multipotent precursori del muscolo scheletrico umano

Generazione di Myospheres Da hESCs di epigenetica Riprogrammazione

Purificazione di progenitori dal muscolo scheletrico

Riepilogo

Esplora Altri Video

Capitoli in questo video

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

Preparazione e la cultura di cellule miogeniche Precursore / mioblasti primari dal muscolo scheletrico di adulti e esseri umani invecchiati

Isolamento dei mioblasti umani, valutazione della differenziazione myogenica e misurazione dell'entrata del calcio a livello di deposito

Isolamento, cultura, caratterizzazione e differenziazione delle cellule del progenitore muscolare umano dalla procedura di biopsia muscolare scheletrica

Isolare cellule staminali da tessuti molli muscoloscheletrici

FACS-isolamento e coltura di progenitori fibro-adipogeni e cellule staminali muscolari da muscolo scheletrico di topo imperturbato e ferito

Identificazione e isolamento di Oligopotent e dei progenitori mieloidi Lineage-commesso dal midollo osseo del topo

Isolamento, Cultura e trapianto di cellule muscolari satellite

Isolamento dei vasi sanguigni derivati ​​da Multipotent precursori del muscolo scheletrico umano

Generazione di Myospheres Da hESCs di epigenetica Riprogrammazione

Isolamento dei vasi sanguigni derivati da Multipotent precursori del muscolo scheletrico umano