We thank the members of the FACS Core Facility in the Department of Biomedicine at Aarhus University for technical support. We thank Alexander Schmitz, the manager of the Mass Cytometry Unit at the Department of Biomedicine, for discussion and support. Scientific Illustrations were created using Biorender.com. This work was funded by an Aarhus Universitets Forskningsfond (AUFF) Starting Grant and a Start Package grant (0071113) from Novo Nordisk Foundation to E.P.

Single-Cell CyTOF Analysis of Myogenic Progenitors in Muscle Regeneration

The authors declare no conflict of interest.

Skeletal muscle regeneration is a dynamic process that relies on the function of adult stem cells. While prior studies have focused on the role of muscle stem cells during regeneration, their progeny in vivo has been understudied, primarily due to a lack of tools to identify and isolate these cell populations15,16,17,18. Here, we present a method to simultaneously identify and isolate ...

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.

<table><tbody><tr><td>15 mL centrifuge tube</td><td>Fisher Scientific</td><td>07-200-886</td><td></td></tr><tr><td>20 G needle</td><td>KDM</td><td>KD-fine 900123</td><td></td></tr><tr><td>28 G, 0.5 mL insulin syringe&amp;nbsp;</td><td>BD</td><td>329461</td><td></td></tr><tr><td>29 G, 0.3 mL insulin syringe</td><td>BD</td><td>324702</td><td></td></tr><tr><td>3 mL syringes</td><td>Terumo medical</td><td>MDSS03SE</td><td></td></tr><tr><td>40 &amp;micro;m cell strainers</td><td>Fisher Scientific</td><td>11587522</td><td></td></tr><tr><td>5 mL polypropylene tubes&amp;nbsp;</td><td>Fisher Scientific</td><td>352002</td><td></td></tr><tr><td>5 mL polystyrene test tubes with 35 &amp;micro;m cell strainer</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>5 mL syringes</td><td>Terumo medical</td><td>SS05LE1</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Fisher Scientific</td><td>05-539-13</td><td></td></tr><tr><td>5-Iodo-2-deoxyuridine (IdU)</td><td>Merck</td><td>I7125-5g</td><td></td></tr><tr><td>anti-CD104 FITC (clone: 346-11A)</td><td>Biolegend</td><td>123605</td><td>Stock = 0.5 mg/mL</td></tr><tr><td>anti-CD11b APC-Cy7 (Clone: M1/70)</td><td>Biolegend</td><td>101226</td><td>Stock = 0.2 mg/mL</td></tr><tr><td>anti-CD31 APC-Cy7 (clone: 390)</td><td>Biolegend</td><td>102440</td><td>Stock = 0.2 mg/mL</td></tr><tr><td>anti-CD45 APC-Cy7 (Clone: 30-F11)</td><td>Biolegend</td><td>103116</td><td>Stock = 0.2 mg/mL</td></tr><tr><td>anti-CD9 APC (clone: KMC8)</td><td>ThermoFisher Scientific</td><td>17-0091-82</td><td>Stock = 0.2 mg/mL</td></tr><tr><td>anti-Sca1 (Ly6A/E) APC-Cy7 (clone: D7)</td><td>Biolegend</td><td>108126</td><td>Stock = 0.2 mg/mL</td></tr><tr><td>anti-&amp;alpha;7 integrin PE (clone: R2F2))</td><td>UBC AbLab</td><td>67-0010-05</td><td>Stock = 1 mg/mL</td></tr><tr><td>BD FACS Aria III (4 laser) instrument</td><td>BD Biosciences</td><td>N/A</td><td>405, 488, 561, and 633 nm laser</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A7030-50G</td><td></td></tr><tr><td>Buprenorphine 0.3 mg/mL</td><td>Ceva</td><td>Vnr 054594</td><td></td></tr><tr><td>CD104 (Clone: 346-11A)</td><td>BD Biosciences</td><td>553745</td><td>Dy162; In-house conjugated</td></tr><tr><td>CD106/VCAM-1 (Clone: 429 MVCAM.A)</td><td>Biolegend</td><td>105701</td><td>Er170; In-house conjugated</td></tr><tr><td>CD11b (Clone: M1/70)</td><td>BD Biosciences</td><td>553308</td><td>Nd148; In-house conjugated</td></tr><tr><td>CD29/Integrin &amp;beta;1 (Clone: 9EG7)</td><td>BD Biosciences</td><td>553715</td><td>Tm169; In-house conjugated</td></tr><tr><td>CD31 (Clone: MEC 13.3)</td><td>BD Biosciences</td><td>557355</td><td>Sm154; In-house conjugated</td></tr><tr><td>CD34 (Clone: RAM34)</td><td>BD Biosciences</td><td>551387</td><td>Lu175; In-house conjugated</td></tr><tr><td>CD44 (Clone: IM7)</td><td>BD Biosciences</td><td>550538</td><td>Yb171; In-house conjugated</td></tr><tr><td>CD45 (Clone: MEC 30-F11)</td><td>BD Biosciences</td><td>550539</td><td>Sm147; In-house conjugated</td></tr><tr><td>CD9 (Clone: KMC8)</td><td>Thermo Fisher Scientific</td><td>14-0091-85</td><td>Yb174; In-house conjugated</td></tr><tr><td>CD90.2/Thy1.2 (Clone: 30-H12)</td><td>BD Biosciences</td><td>553009</td><td>Nd144; In-house conjugated</td></tr><tr><td>CD98 (Clone: H202-141)</td><td>BD Biosciences</td><td>557479</td><td>Pr141; In-house conjugated</td></tr><tr><td>Cell Acquisition Solution/Maxpar CAS-buffer</td><td>Standard Biotools</td><td>201240</td><td></td></tr><tr><td>Cell-ID Intercalator-Iridium</td><td>Standard Biotools</td><td>201192B</td><td>cationic nucleic acid intercalator</td></tr><tr><td>Cisplatin</td><td>Merck</td><td>P4394</td><td>Pt195</td></tr><tr><td>Cisplatin (cis-Diammineplatinum(II) dichloride)</td><td>Merck</td><td>P4394</td><td></td></tr><tr><td>Clear 1.5 mL tube</td><td>Fisher Scientific</td><td>11926955</td><td></td></tr><tr><td>Collagenase, Type II</td><td>Worthington Biochemical Corporation</td><td>LS004177</td><td></td></tr><tr><td>Counting chamber</td><td>Merck</td><td>BR718620-1EA</td><td></td></tr><tr><td>CXCR4/SDF1 (Clone: 2B11/CXCR4 )</td><td>BD Biosciences</td><td>551852</td><td>Gd158; In-house conjugated</td></tr><tr><td>DAPI (1 mg/mL)</td><td>BD Biosciences</td><td>564907</td><td></td></tr><tr><td>Dark 1.5 mL tube</td><td>Fisher Scientific</td><td>15386548</td><td></td></tr><tr><td>Dispase II</td><td>Thermo Fisher Scientific</td><td>17105041</td><td></td></tr><tr><td>Dissection Scissors</td><td>Fine Science Tools</td><td>14568-09</td><td></td></tr><tr><td>DMEM (low glucose, with pyruvate)</td><td>Thermo Fisher Scientific</td><td>11885-092</td><td></td></tr><tr><td>EDTA (Ethylenediaminetetraacetic acid disodium salt)</td><td>Merck</td><td>E5134</td><td>Na&lt;sub&gt;2&lt;/sub&gt;EDTA-2H&lt;sub&gt;2&lt;/sub&gt;0</td></tr><tr><td>EQ Four Element Calibration Beads (EQ beads)</td><td>Standard Biotools</td><td>201078</td><td>Calibration beads</td></tr><tr><td>Fetal Bovine Serum, qualified, Brazil origin</td><td>Thermo Fisher Scientific</td><td>10270106</td><td></td></tr><tr><td>Forceps Dumont #5SF</td><td>Fine Science Tools</td><td>11252-00</td><td></td></tr><tr><td>Forceps Dumont #7</td><td>Hounisen.com</td><td>1606.3350</td><td></td></tr><tr><td>Goat serum</td><td>Thermo Fisher Scientific</td><td>16210-072</td><td></td></tr><tr><td>Helios CyTOF system</td><td>Standard Biotools</td><td>N/A</td><td></td></tr><tr><td>Horse Serum, heat inactivated, New Zealand origin</td><td>Thermo Fisher Scientific</td><td>26-050-088</td><td></td></tr><tr><td>IdU</td><td>Merck</td><td>I7125</td><td>I127</td></tr><tr><td>Iridium-Intercalator</td><td>Standard Biotools</td><td>201240</td><td>Ir191/193</td></tr><tr><td>Isoflurane/Attane Vet</td><td>ScanVet</td><td>Vnr 055226</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>M/3900/17</td><td></td></tr><tr><td>Myf5 (Clone: C-20)</td><td>Santa Cruz Biotechnology</td><td>Sc-302</td><td>Yb173; In-house conjugated</td></tr><tr><td>MyoD (Clone: 5.8A)</td><td>BD Biosciences</td><td>554130</td><td>Dy164; In-house conjugated</td></tr><tr><td>MyoG (Clone: F5D)</td><td>BD Biosciences</td><td>556358</td><td>Gd160; In-house conjugated</td></tr><tr><td>Nalgene Rapid-Flow Sterile Disposable Bottle Top 0.20 &amp;mu;M PES Filters</td><td>Thermo Fisher Scientific</td><td>595-4520</td><td></td></tr><tr><td>Notexin</td><td>Latoxan</td><td>L8104</td><td>Resuspend to 50 &amp;micro;g/ml in sterile PBS. Keep stocks (e.g. 50 &amp;micro;l) at -20 &amp;deg;C</td></tr><tr><td>Nutrient mixture F-10 (Ham&#039;s)</td><td>Thermo Fisher Scientific</td><td>31550031</td><td></td></tr><tr><td>pAkt (Clone: D9E)</td><td>Standard Biotools</td><td>3152005A</td><td>Sm152</td></tr><tr><td>Pax7 (Clone: PAX7)</td><td>Santa Cruz Biotechnology</td><td>Sc-81648</td><td>Eu153; In-house conjugated</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL) (Pen/Strep)</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>PES Filter Units 0.20 &amp;mu;M</td><td>Fisher Scientific</td><td>15913307</td><td></td></tr><tr><td>PES Syringe Filter</td><td>Fisher Scientific</td><td>15206869</td><td></td></tr><tr><td>Petri dish</td><td>Sarstedt</td><td>82.1472.001</td><td></td></tr><tr><td>PFA 16% EM grade</td><td>MP Biomedicals</td><td>219998320</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Fisher Scientific</td><td>10375810</td><td></td></tr><tr><td>Potassium phosphate, monobasic,&amp;nbsp;anhydrous (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>10573181</td><td></td></tr><tr><td>pRb (Clone: J112-906)</td><td>Standard Biotools</td><td>3166011A</td><td>Er166</td></tr><tr><td>pS6 kinase (Clone: N7-548)</td><td>Standard Biotools</td><td>3172008A</td><td>Yb172</td></tr><tr><td>Sca-1 (Clone: E13-161.7)</td><td>BD Biosciences</td><td>553333</td><td>Nd142; In-house conjugated</td></tr><tr><td>Sodium Azide</td><td>Sigma Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>10553515</td><td></td></tr><tr><td>Sodium phosphate, dibasic, heptahydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;-6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Merck</td><td>S9390</td><td></td></tr><tr><td>Sterile saline solution 0.9%</td><td>Fresenius</td><td>B306414/02</td><td></td></tr><tr><td>&amp;alpha;7 integrin (Clone: 3C12)</td><td>MBL international</td><td>K0046-3</td><td>Ho165; In-house conjugated</td></tr></tbody></table>

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.

Here we present an overview of the experimental setup for using this combined approach which includes (i) high-dimensional CyTOF analysis of an acute injury time course by notexin injection to study the cellular and molecular dynamics of stem and progenitor cells in skeletal muscle (Figure 1, top scheme); and (ii) FACS of stem and progenitor cells using two cell surface markers, CD9 and CD104, to isolate these populations and perform in-depth studies of their function (F...

Read this Scientific Article Protocol about Author Spotlight: Investigating Cellular and Molecular Dynamics during Muscle Regeneration Using Cutting-Edge Single-Cell Technologies at JoVE.com

Author Spotlight: Investigating Cellular and Molecular Dynamics During Muscle Regeneration Using Cutting-Edge Single-Cell Technologies

The protocol presented here enables the identification and high-dimensional analysis of muscle stem and progenitor cells by single-cell mass cytometry and their purification by FACS for in-depth studies of their function. This approach can be applied to study regeneration dynamics in disease models and test the efficacy of pharmacological interventions.

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 ...

identification-analysis-myogenic-progenitors-vivo-during-acute

Research

JoVE Journal

Biology

955 Views.  Aarhus University. Ons onderzoek onderzoekt de moleculaire mechanismen die ten grondslag liggen aan stamceldisfunctie bij veroudering en ziekte. Ons doel is om te begrijpen hoe spierstam- en voorlopercellen interageren met andere cellen in de spiermicro-omgeving om effectieve regeneratie te bereiken. We onderzoeken ook of een kale celcommunicatie de spierstamcelfunctie beïnvloedt bij veroudering en ziekte.Een belangrijke barrière voor het onderzoeken van cel-celcommuni...

Video: Auteur in de schijnwerpers: onderzoek naar cellulaire en moleculaire dynamica tijdens spierregeneratie met behulp van geavanceerde single-cell-technologieën

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.

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

Developmental Biology

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

Genetics

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.

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

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

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.

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

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

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

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

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

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

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

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

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

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

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

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

Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry

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