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Description: Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle ...

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A correction was made to <a href="http://www.jove.com/Details.php?ID=1246">Chris's testing Test 3/26/09 test test testChris's testing Test 3/26/09 test test testChris's testing Test 3/26/09 test t12</a>. There was an error with an author's name. The author's last name had a typo and was corrected to: Nima Saeidi instead of: Nima Saedi.

Erratum: Chris's testing Test 3/26/09 test test testChris's testing Test 3/26/09 test test testChris's testing Test 3/26/09 test t12

Skeletal muscle constitutes the largest tissue by mass in the body and regulates multiple functions, from eyesight to respiration, from posture to movement, as well as metabolism1. Therefore, maintaining skeletal muscle integrity and function is critical to health. Skeletal muscle tissue, which consists of tightly packed bundles of multinucleated myofibers surrounded by a complex network of nerves and blood vessels, exhibits remarkable regenerative potential1,2.
The main drivers of skeletal muscle regeneration are adult muscle stem cells (MuSCs). Also known as satellite cells, due to their unique anatomical location adjacent to the plasma membrane of the myofiber and beneath the basal lamina, they were first identified in 19613. MuSCs express a unique molecular marker, the transcription factor paired box 7 (Pax7)4. Mostly quiescent in healthy adults, they become activated upon muscle injury and proliferate to give rise to progeny that will (i) differentiate into fusion-competent muscle cells that will form new myofibers to repair muscle damage or (ii) self-renew to replenish the stem cell pool5.
At the cellular and molecular level, the process of regeneration is quite dynamic and involves cell-state transitions, characterized by the coordinated expression of key myogenic transcription factors, also known as myogenic regulatory factors (MRFs)6,7. Prior in vivo developmental studies, lineage tracing experiments, and cell culture work using myoblasts&#160;have shown that sequential expression of these transcription factors drives myogenesis, with myogenic factor 5 (Myf5) being expressed upon activation, myogenic differentiation 1 (MyoD1) expression marking commitment to the myogenic program, and myogenin (MyoG) expression marking differentiation8,9,10,11,12,13,14. Despite this knowledge and the discovery of cell surface markers to purify MuSCs, strategies and tools to identify and isolate discrete populations along the myogenic differentiation path and resolve a myogenic progression in vivo have been lacking15,16,17,18.
Here, we present a novel method, based on recently published research, which enables the identification of stem and progenitor cells in skeletal muscle and the analysis of their cellular, molecular, and proliferation dynamics in the context of acute muscle injury19. This approach relies on single-cell mass cytometry (also known as Cytometry by Time of Flight [CyTOF]) to simultaneously detect key cell surface markers (&#945;7&#160;integrin, CD9, CD44, CD98, and CD104), intracellular myogenic transcription factors (Pax7, Myf5, MyoD, and MyoG) and a nucleoside analog (5-Iodo-2&#8242;-deoxyuridine, IdU), to monitor cells in S phase19,20,21,22,23. Moreover, the protocol presents a strategy based on the detection of two cell surface markers, CD9 and CD104, to purify these cell populations by fluorescence-activated cell sorting (FACS), therefore enabling future in-depth studies of their function in the context of injury and muscle diseases. While primary myoblasts have been extensively used in the past to study the late stages of myogenic differentiation in vitro, it is not known whether they recapitulate the molecular state of muscle progenitor cells found in vivo24,25,26,27,28,29,30. The production of myoblasts is laborious and time-consuming, and the molecular state of this primary culture changes rapidly upon passaging31. Hence, freshly isolated myogenic progenitors purified with this method will provide a more physiological system to study myogenesis and the effect of genetic or pharmacological manipulations ex-vivo.
The protocol presented here can be applied to address a variety of research questions, for example, to study the dynamics of the myogenic compartment in vivo in animal models of muscle diseases, in response to acute genetic manipulations or upon pharmacological interventions, therefore deepening our understanding of muscle stem cell dysfunction in different biological contexts and facilitating the development of novel therapeutic interventions.

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identification and analysis of myogenic progenitors in vivo during acute skeletal muscle injury by high-dimensional single-cell mass cytometry

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle stem cells become activated upon muscle injury. Following activation, they proliferate, and most of their progeny differentiate to generate fusion-competent muscle cells while the remaining self-renews to replenish the stem cell pool. While the identity of muscle stem cells was defined more than a decade ago, based on the co-expression of cell surface markers, myogenic progenitors were identified only recently using high-dimensional single-cell approaches. Here, we present a single-cell mass cytometry (cytometry by time of flight [CyTOF]) method to analyze stem cells and progenitor cells in acute muscle injury to resolve the cellular and molecular dynamics that unfold during muscle regeneration. This approach is based on the simultaneous detection of novel cell surface markers and key myogenic transcription factors whose dynamic expression enables the identification of activated stem cells and progenitor cell populations that represent landmarks of myogenesis. Importantly, a sorting strategy based on detecting cell surface markers CD9 and CD104 is described, enabling prospective isolation of muscle stem and progenitor cells using fluorescence-activated cell sorting (FACS) for in-depth studies of their function. Muscle progenitor cells provide a critical missing link to study the control of muscle stem cell fate, identify novel therapeutic targets for muscle diseases, and develop cell therapy applications for regenerative medicine. The approach presented here can be applied to study muscle stem and progenitor cells in vivo in response to perturbations, such as pharmacological interventions targeting specific signaling pathways. It can also be used to investigate the dynamics of muscle stem and progenitor cells in animal models of muscle diseases, advancing our understanding of stem cell diseases and accelerating the development of therapies.

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Il saggio di migrazione dei neutrofili transuroepithelial consente la valutazione quantitativa delle migrazioni PMN umani in coltura vescica attraverso strati di cellule epiteliali in risposta a vari stimoli (Figura 1B). Mentre il protocollo Ã¨ semplice, ci sono una serie di variabili che possono influenzare la migrazione di PMN e conseguentemente influenzare la riproducibilitÃ  di questo test. Le misure dovrebbero essere prese durante la preparazione dei supporti permeabili e la PMN per ridurre la variabilitÃ  tra i replicati tecnici e biologici. Per esempio, permeabile solo supporta contenente sufficientemente confluenti 5637 strati cellulari devono essere utilizzati in un esperimento. Confluenza delle cellule 5637 Ã¨ valutato attraverso un saggio funzionale che misura impermeabilitÃ  ai liquidi. Se medie aggiunto ai equilibra bacino superiore attraverso il supporto permeabile, allora le cellule 5637 non sono sufficientemente confluenti per condurre l&#39;esperimento. Se il volume nel serbatoio superiore Ã¨ mantenuta,poi il supporto permeabile puÃ² essere utilizzato per valutare la migrazione PMN. Abbiamo misurato resistenza elettrica transepiteliale in questo sistema, che si erge su modestamente confluenza delle cellule, se si sceglie questo metodo, occorre prestare attenzione a non contaminare la configurazione altrimenti sterile. Confluenza delle cellule 5637 7 giorni dopo la semina puÃ² essere influenzata da molteplici fattori, incluso il numero passaggi di cellule e il numero di cellule inseminate sul supporto permeabile. Inoltre, la quantitÃ  di tempo che le cellule vengono incubate 5637 sul supporto permeabile in posizione capovolta durante la semina non deve superare 16 ore (Figura 1A). Per la riproducibilitÃ  ottimale, il protocollo deve essere rigorosamente. Infine, permeabile supporta contenente confluenti 5637 strati di cellule devono essere utilizzati entro 1-2 giorni, e le membrane dei supporti non deve mai essere toccato durante sia la crescita delle cellule del 5637 o durante il saggio di migrazione dei neutrofili transuroepithelial. Oltre alle cellule 5637, variabilitÃ  puÃ² anche essere introdotto durante la preparazione PMN. Utilizzando il protocollo dettagliato sopra per isolare PMN, 1 ml di sangue umano produce tipicamente di circa 10 6 PMN, anche se questo numero varia da individuo a individuo. Una volta che la resa tipica di sangue di un singolo donatore Ã¨ noto, il protocollo di isolamento puÃ² essere scalata verso l&#39;alto o verso il basso di conseguenza. PMN da individui sani o malati deve essere evitato, e diversi donatori PMN deve essere utilizzato per biologico replica per garantire che i risultati osservati sono riproducibili. PMN deve essere maneggiato con delicatezza e sterilmente evitare azionamenti durante l&#39;isolamento. Infine, la tempistica delle procedure sperimentali Ã¨ di fondamentale importanza, come PMN non sopravvivono per lunghi periodi di tempo, una volta rimossi dal corpo. Utilizziamo PMN entro 1 ora di portare a termine la procedura di isolamento. Alla luce di queste considerazioni, almeno 3 tecnici repliche dovrebbero essere inclusi in ogni replica biologica. Il numero dei PMN nel serbatoio inferiore dopo 1 h Ã¨ mostrato in Figura 2 normalizzata a 10 6 ingresso PMN. In alternativa, i numeri PMN puÃ² essere paragonato a un controllo interno dopo la normalizzazione di ingresso PMN, che puÃ² ridurre la variabilitÃ  tra biologico replica. L&#39;adesione al protocollo descritto sopra con attenzione ai dettagli consente l&#39;enumerazione di migrazione dei PMN in risposta a stimoli tra cui i batteri (Figura 2A) e sostanze chemoattractant (Figura 2B). <img src="/files/ftp_upload/50919/50919fig1.jpg" alt="Figura 1" fo:content-width="5in" fo:hires="/img/sprite.png?v=68"/> Figura 1. Schematica di disegno sperimentale. (A) 5637 cellule epiteliali della vescica sono seminate su supporti permeabili invertiti, i supporti sono raddrizzati in una piastra da 24 pozzetti e le cellule sono cresciute a confluenza. (B) Permeabile supporte contenenti 5637 cellule confluenti sono invertiti e infettati con E. coli sul lato apicale degli strati epiteliali. Alternativamente, chemoattractants possono essere collocati nel serbatoio inferiore. I supporti permeabili sono raddrizzati in una piastra bassa attaccamento e PMN umani appena isolate sono applicate al serbatoio superiore (che rappresenta il lato basolaterale degli strati epiteliali). PMN migrare attraverso l&#39;epitelio e vengono enumerati dal serbatoio inferiore utilizzando un emocitometro. <img src="/files/ftp_upload/50919/50919fig2.jpg" alt="Figura 2" fo:content-width="2.5in" fo:src="/files/ftp_upload/50919/50919fig2highres.jpg"/> Figura 2. PMN migrare attraverso epiteli vescica in risposta a vari stimoli. (A) Infezione da non patogeno E. coli ceppo MG1655, MG1655 calore uccise (HKMG) o UPEC mutante UTI89 ybcL :: gatto suscita migrazione significativamente piÃ¹ PMN di mock infezione o infezione con wild-type UPEC ceppo UTI89, una cistite isolano (*, P &lt;0,001). (B) L&#39;aggiunta di fMLF (100 nM) o IL-8 (100 ng / ml) per i risultati serbatoio inferiore a migrazione significativamente piÃ¹ PMN che non il trattamento finto (*, P &lt;0,001). I dati rappresentano la media e la deviazione standard di almeno 3 replicati biologici. Differenze statisticamente significative sono state determinate utilizzando il test t di Student uno spaiato.

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Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle ...