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.

Animal procedures were approved by the Danish animal experiments inspectorate (protocol # 2022-15-0201-01293), and experiments were performed in compliance with the institutional guidelines of Aarhus University. Analgesia (buprenorphine) is provided in drinking water 24 h prior to injury for the mice to adapt to the taste. Supplying buprenorphine in drinking water is continued for 24 h post-injury. Together with a subcutaneous (s.c.) injection of buprenorphine at the time of acute muscle injury, buprenorphine in the drin...

Single-Cell CyTOF Analysis of Myogenic Progenitors in Muscle Regeneration

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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 &#181;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>11587522</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polypropylene tubes&#160;</td>
			<td>Fisher Scientific</td>
			<td>352002</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polystyrene test tubes with 35 &#181;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-&#945;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 &#946;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>Na2EDTA-2H20</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 &#956;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 &#181;g/ml in sterile PBS. Keep stocks (e.g. 50 &#181;l) at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Nutrient mixture F-10 (Ham&#39;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 &#956;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,&#160;anhydrous (KH2PO4)</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 (Na2HPO4-6H2O)</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>&#945;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.

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

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

Our research investigates the molecular mechanisms underlying stem cell dysfunction in aging and disease. Our goal is to understand how muscle stem and progenitor cells interact with other cells in the muscle microenvironment to achieve effective regeneration. We are also studying whether a barren cell communication impacts muscle stem cell function in aging and disease.A major barrier to investigating cell-cell communication within the stem cell niche has been the lack of technologies that provide single cell resolution. Single cell mass cytometry allows simultaneous high throughput quantitative analysis on multiple cell types and molecular phenotypes in complex tissues. Our protocol enables purification and deep profiling of rare stem cells and the progeny during muscle regeneration using antibodies to cell surface markers and myogenic transcription factors.Studying these cells helps us understand how muscle stem cell fates is controlled, and uncover the networks that drive cell state transitions doing effective regeneration. Measuring up to 50 cell surface and intracellular protein simultaneously at a single cell level is a valuable approach for investigating cellular function in muscle tissue. This technique can identify unique molecular signatures and reveal novel mechanisms of muscle stem cell dysfunction in aging and disease.This protocol allows to investigate how cell state transition impact tissue regeneration in health and disease. The signature of activated stem cells we present here enable studies of stem cell quiescence. They were not previously possible.We can now purify activated stem cells, CD98 High, CD44 High, based solely on cell surface marker, and study for the first time, how they return to quiescence.

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

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.

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