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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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PMN ×¦×¨×™×š ×œ×”×™×•×ª ×ž×˜×•×¤×œ ×‘×¢×“×™× ×•×ª ×•×‘sterilely ×›×“×™ ×œ×”×™×ž× ×¢ ×ž×”×¤×¢×œ×” ×‘×‘×™×“×•×“. ×œ×‘×¡×•×£, ×”×¢×™×ª×•×™ ×©×œ ×”×¤×¨×•×¦×“×•×¨×•×ª ×—×™×•× ×™, ×›×ž×• PMN ×œ× ×œ×©×¨×•×“ ×‘×ž×©×š ×¤×¨×§×™ ×–×ž×Ÿ ××¨×•×›×™× ×”×•×¡×¨×• ×¤×¢× ××—×ª ×ž×”×’×•×£. ×× ×• ×ž× ×¦×œ×™× PMN ×‘×ª×•×š ×©×¢×” 1 ×©×œ ×”×©×œ×ž×ª ×”×œ×™×š ×”×‘×™×“×•×“. ×‘×”×ª×—×©×‘ ×‘×©×™×§×•×œ×™× ××œ×”, ×œ×¤×—×•×ª 3 ×˜×›× ×™×™× ×ž×©×›×¤×œ ×¦×¨×™×›×™× ×œ×”×™×•×ª ×›×œ×•×œ×™× ×‘×›×œ ××—×“ ×œ×©×›×¤×œ ×‘×™×•×œ×•×’×™×™×. ×ž×¡×¤×¨ PMN ×‘×ž××’×¨ ×”×ª×—×ª×•×Ÿ ×œ××—×¨ ×©×¢×” 1 ×ž×•×¦×’ ×‘××™×•×¨ 2 ×ž× ×•×¨×ž×œ×™× ×¢×“ 10 PMN ×§×œ×˜ 6. ×œ×—×œ×•×¤×™×Ÿ, × ×™×ª×Ÿ ×œ×”×©×•×•×ª ×ž×¡×¤×¨×™ PMN ×œ×‘×§×¨×” ×¤× ×™×ž×™×ª ×œ××—×¨ × ×•×¨×ž×œ×™×–×¦×™×” ×œ×§×œ×˜ PMN, ××©×¨ ×¢×©×•×™ ×œ×”×¤×—×™×ª ×•×¨×™××¦×™×” ×‘×™×Ÿ ×ž×©×›×¤×œ ×‘×™×•×œ×•×’×™. ×”×™×¦×ž×“×•×ª ×œ×¤×¨×•×˜×•×§×•×œ ×©×ª×•××¨ ×œ×¢×™×œ ×¢× ×ª×©×•×ž×ª ×œ×‘ ×œ×¤×¨×˜×™× ×ž××¤×©×¨×ª ×¡×¤×™×¨×” ×©×œ ×”×’×™×¨×ª PMN ×‘×ª×’×•×‘×” ×œ×’×™×¨×•×™×™×, ×›×•×œ×œ ×—×™×™×“×§×™× (××™×•×¨ 2 ×) ×•×—×•×ž×¨×™ chemoattractant (××™×•×¨ 2). <img src="/files/ftp_upload/50919/50919fig1.jpg" alt="××™×•×¨ 1" fo:content-width="5in" fo:hires="/img/sprite.png?v=68"/> ××™×•×¨ 1. ×¡×›×ž×˜×™ ×©×œ ×¢×™×¦×•×‘ × ×™×¡×™×•× ×™. () 5637 ×ª××™ ××¤×™×ª×œ ×©×œ×¤×•×—×™×ª â€‹â€‹×”×©×ª×Ÿ ×”× ×–×•×¨×¢×™× ×¢×œ ×ª×•×ž×š ×—×“×™×¨ ×”×¤×•×š, ×ª×•×ž×š ×”× ×™×™×©×¨×• ×œ×ª×•×š ×¦×œ×—×ª 24 ×’×, ×•××ª ×”×ª××™× ×’×“×œ×™× ×œ× ×§×•×“×ª ×ž×¤×’×©. (×‘) ×—×“×™×¨ sup×™×¦×™××•×ª ×”×ž×›×™×œ×•×ª 5637 ×ª××™ ×ž×—×•×‘×¨×•×ª ×”× ×”×¤×•×›×™× ×•× ×’×•×¢×™× E. coli ×‘×¦×“ ×”×¤×¡×’×” ×©×œ ×©×›×‘×•×ª ××¤×™×ª×œ. ×œ×—×œ×•×¤×™×Ÿ, × ×™×ª×Ÿ ×œ×”×¦×™×‘ chemoattractants ×‘×ž××’×¨ ×”×ª×—×ª×•×Ÿ. ×ª×•×ž×š ×—×“×™×¨ ×”× ×™×™×©×¨×• ×œ×ª×•×š ×¦×œ×—×ª × ×ž×•×›×” ×ž×¦×•×¨×£, ×•PMN ××“× ×ž×‘×•×“×“ ×˜×¨×™ ×ž×•×—×œ×™× ×¢×œ ×”×ž××’×¨ ×”×¢×œ×™×•×Ÿ (×”×ž×™×™×¦×’ ××ª ×¦×“ basolateral ×©×œ ×©×›×‘×•×ª ××¤×™×ª×œ). PMN × ×•×“×“ ×¢×œ ×¤× ×™ ×”××¤×™×ª×œ ×•×”× ×ž× ×• ×ž×”×ž××’×¨ ×”×ª×—×ª×•×Ÿ ×‘××ž×¦×¢×•×ª hemacytometer. <img src="/files/ftp_upload/50919/50919fig2.jpg" alt="××™×•×¨ 2" fo:content-width="2.5in" fo:src="/files/ftp_upload/50919/50919fig2highres.jpg"/> ××™×•×¨ 2. 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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 ...