We are indebted to Clemire Cimper for her excellent technical support. Work in the laboratory of H.L. was funded by Institut Pasteur, Centre National pour la Recherche Scientific, and the Agence Nationale de la Recherche (Laboratoire d&#39;Excellence Revive, Investissement d&#39;Avenir; ANR-10-LABX- 73), the Agence Nationale de la Recherche (ANR-16-CE13-0017-01) and Fondation ARC (PJA 20161205028). C.C. and A.C. are funded by the Ph.D. and postdoctoral fellowships from the Revive Consortium.

Animals were handled as per European Community guidelines and the ethics committee of the Institut Pasteur (CETEA) approved protocols.
1. Preparations of the Stock Solutions
<ol>
	<li>Prepare the materials for muscle sample fixation.Dissolve 0.5 g of tragacanth gum with 20 mL water at RT to make the freezing-embedding medium for muscle fixation.</li>
	<li>Prepare the solutions for SA-&#946;-Gal staining.
	<ol>
		<li>Prepare the stock solutions of K3Fe(CN)6</s...

<ol>
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(119), (2017).</li><li>Le Roux, I., Konge, J., Le Cam, L., Flamant, P., Tajbakhsh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numb+is+required+to+prevent+p53-dependent+senescence+following+skeletal+muscle+injury.">Numb is required to prevent p53-dependent senescence following skeletal muscle injury.</a> Nat Commun. 6, 8528 (2015).</li><li>Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+to+detect+senescence-associated+beta-galactosidase+(SA-betagal)+activity,+a+biomarker+of+senescent+cells+in+culture+and+in+vivo.">Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo.</a> Nat Protoc. 4 (12), 1798-1806 (2009).</li><li>Dimri, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomarker+that+identifies+senescent+human+cells+in+culture+and+in+aging+skin+in+vivo.">A biomarker that identifies senescent human cells in culture and in aging skin in vivo.</a> Proc Natl Acad Sci U S A. 92 (20), 9363-9367 (1995).</li><li>Lee, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+beta-galactosidase+is+lysosomal+beta-galactosidase.">Senescence-associated beta-galactosidase is lysosomal beta-galactosidase.</a> Aging Cell. 5 (2), 187-195 (2006).</li><li>Krishna, D. R., Sperker, B., Fritz, P., Klotz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+pH+6+beta-galactosidase+activity+indicate+cell+senescence?.">Does pH 6 beta-galactosidase activity indicate cell senescence?.</a> Mech Ageing Dev. 109 (2), 113-123 (1999).</li><li>Cristofalo, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SA+beta+Gal+staining:+biomarker+or+delusion.">SA beta Gal staining: biomarker or delusion.</a> Exp Gerontol. 40 (10), 836-838 (2005).</li></ol>

Here, we present a method to detect both senescent and pluripotent stem cells in the skeletal muscle of reprogrammable mice. This method could be used to evaluate and quantify both senescence and induce cellular plasticity in vivo, and examine the role of senescence in tissue repair and regeneration.
In the current protocol, the senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is used to detect in vivo senescent cells in the skeletal muscle. This assay detects...

Cellular senescence is a form of stress response characterized by a stable cell-cycle arrest. In the last decade, research has firmly established that senescence is associated with various biological and pathological processes including embryonic development, fibrosis, and organism ageing1,2. Cellular senescence was first identified in human fibroblasts at the end of their replicative lifespan triggered by telomere shortening3. Besides replicative stress, there are many other stimuli that can induce senescence, including DNA damage, oxidative stress, oncogenic signals, and genomic/epigenomic alterations, any of which may eventually activate the p53/p21 and/or pRB pathways to establish and reinforce the permanent growth arrest1. One of the important characteristics of senescent cells is that they remain metabolically active and robustly express a senescence-associated secretory phenotype (SASP): secretion of many inflammatory cytokines, growth factors, and extracellular matrix factors4. SASP factors have been proposed to play an important role in mediating and amplifying the senescence effect, due to their potent effects on attracting immune cells and altering local and systemic tissue milieus1. Interestingly, senescence has been recently proposed to be important for tissue repair and regeneration5,6. In addition, data from several labs, including ours, has suggested that tissue damage-induced senescence might enhance cellular plasticity, via SASPs, to promote regeneration7-9. Therefore, all the emerging data highlight the importance of studying senescence in vivo.
In the post induced pluripotent stem cell&#160;(iPSC) era, cellular plasticity is the capacity of a cell to acquire a new identity and to adopt an alternative fate when exposed to different stimuli both in culture and in vivo10. It is known that full reprogramming can be achieved in vivo11,12, where the expression of the the cassette containing four Yamanaka factors: Oct4, Sox2, Klf4, and c-Myc (OSKM) can be induced in vivo to promote teratomas formation in multiple organs. Therefore, a reprogrammable mouse model (i4F) can be used as a powerful system to identify critical regulators and pathways that are important for cellular plasticity11.
A suitable and sensitive in vivo system is essential to understand how cellular senescence regulates cellular plasticity in the context of tissue regeneration. Here, we present a robust system and a detailed protocol to evaluate the link between senescence and cellular plasticity in the context of tissue regeneration. The combination of cardiotoxin (CTX) induced muscle damage in the Tibialis Anterior (TA) muscle group, a well-established system to study tissue regeneration, and the i4F mouse model, allows the detection of both cellular senescence and in vivo reprogramming during muscle regeneration.
To evaluate the link between cellular plasticity and senescence, i4F mice are injured with CTX to induce acute muscle damage and treated with doxycycline (0.2 mg/mL) over 7 days to induce in vivo reprogramming. While a CTX induced acute muscle damage and regeneration protocol has been recently published13, for ethical reasons, this procedure will be omitted in the current protocol. TA muscle samples will be collected at 10 days post injury13, when the peak of senescent cells have been previously observed14. Here, this detailed protocol describes all the steps required to evaluate the level of senescence (via SA-&#946;-Gal) and reprogramming (via IHC staining of Nanog).
Senescence-associated beta-galactosidase (SA-&#946;-Gal) assay is the most commonly used assay to detect senescent cells both in culture and in vivo15. Compared to other assays, the SA-&#946;-Gal assay allows the identification of the senescent cells in their native environment with intact tissue architecture, which is particularly important for in vivo study. Moreover, it is possible to couple the SA-&#946;-Gal assay with other markers using IHC. However, the SA-&#946;-Gal assay does require fresh or frozen samples, which remains a major limitation. When fresh or frozen tissues are routinely available, such as frozen TA muscle samples, SA-&#946;-Gal is obviously the most suitable assay to detect senescent cells. Nanog is the marker used to detect reprogramed cells for two reasons: 1) it is an essential marker for pluripotency; 2) more importantly, its expression is not driven by doxycycline (dox), therefore it detects induced pluripotency rather than the forced expression of the Yamanaka cassette.
It is important to note, the staining protocols presented in this study can be conducted separately to simplify the quantification procedure, but can also be done in a co-staining procedure to visualize both senescent and pluripotent stem cells on the same section.

<table>
	<tbody>
		<tr>
			<td>K3Fe(CN)6</td>
			<td>Sigma</td>
			<td>13746-66-2</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>K4Fe(CN)6</td>
			<td>Sigma</td>
			<td>14459-95-1</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>7786-30-3</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Sigma</td>
			<td>B4252</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma</td>
			<td>D3447</td>
			<td>For inducing in vivo reprogramming</td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Lotaxan Valence, France</td>
			<td>L8102</td>
			<td>For muscle injury</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>111-30-8</td>
			<td>For Fixation solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron microscopy science</td>
			<td>50-980-487</td>
			<td>For Fixation solution</td>
		</tr>
		<tr>
			<td>NaCitrate : Sodium Citrate monobasic bioxtra, anhydre</td>
			<td>Sigma</td>
			<td>18996-35-5</td>
			<td>For permeabilization solution</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Sigma</td>
			<td>93443</td>
			<td>For permeabilization solution</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A3608</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>Antibody anti- Nanog</td>
			<td>Cell signalling</td>
			<td>8822S</td>
			<td>Rabbit monoclonal antibody</td>
		</tr>
		<tr>
			<td>EnVision+ Kits (HRP. Rabbit. DAB+)</td>
			<td>Dako</td>
			<td>K4010</td>
			<td>For Nanog revelation</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Leica</td>
			<td>380159EOF</td>
			<td>Counterstainning</td>
		</tr>
		<tr>
			<td>Fast red</td>
			<td>Vector Laboratories</td>
			<td>H-3403</td>
			<td>Counterstainning</td>
		</tr>
		<tr>
			<td>Thermo Scientific Shandon Immu-Mount</td>
			<td>Fisher scientific</td>
			<td>9990402</td>
			<td>Mounting solution</td>
		</tr>
		<tr>
			<td>Quick-hardening mounting medium for microscopy : Eukitt&#174;</td>
			<td>Sigma</td>
			<td>25608-33-7</td>
			<td>Mounting solution</td>
		</tr>
		<tr>
			<td>Microscope Phase Contrast Brightfield CKX41: 10X-20X-40X objectives</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td>Microscope for Nanog quantification</td>
		</tr>
		<tr>
			<td>Mouse: i4F-A</td>
			<td>Abad et al., 2013</td>
			<td>N/A</td>
			<td>Reprogrammable mouse model</td>
		</tr>
		<tr>
			<td>Skeletal muscle, Tibialis Anterior</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Scanner</td>
			<td>Zeiss</td>
			<td>Axio Scan Z1</td>
			<td>slides scanning</td>
		</tr>
	</tbody>
</table>

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.

Detecting muscle injury-induced cellular senescence
It has been recently demonstrated that muscle injury induces transient cellular senescence14. At 10 days post-injury (DPI), the majority of the damaged myofibers are undergoing regeneration with centrally located nuclei, a hallmark of regenerating myofibers, and the architecture of the muscle is re-established. The infiltrating inflammatory cells are dramat...

Watch this Scientific Journal Video about Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle at JoVE.com

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.

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 ...

The overall goal of this protocol is to evaluate both in vivo senescence and reprogramming in the skeletal muscle upon tissue injury. This method can help to address critical questions in the regenerative medicine field, such as how to enhance cellular plasticity to promote muscle regeneration. The main advantage of this technique is to provide a reliable method to detect and quantify senescence cells in the resident environment.Cell senescence has been recently implicated in tissue repair and regeneration. Understanding the mechanisms of how senescence contributes to tissue repair and the regeneration will certainly have an impact on regenerative medicine. We first had the idea for this method when we investigated the potential impact of tissue damage on in vivo reprogramming in skeletal muscle.To begin this procedure, fix the slides of muscle samples in 1%paraformaldehyde and 0.2%glutaraldehyde in PBS for four minutes. Then, wash the slides with PBS two times, each time for 10 minutes. Then, wash the slides with PBS, pH 5.5 for 30 minutes.Next, incubate the sections with the X-gal solution in the dark at 37 degrees Celsius for a minimum of 24 hours. Afterward, wash the slides with PBS three times, each time for 10 minutes. Then, post-fix the slides with PBS containing 1%paraformaldehyde for 30 minutes.Finally, wash the slides with PBS three times. Subsequently, counterstain with 0.2%eosin by immersing the slides in the eosin solution for one minute. Then, rinse them with distilled water briefly.Mount the slides with aqueous non-fluorescing mounting medium. Fix the slides with PBS containing 4%paraformaldehyde for 10 minutes. Wash them with PBS twice, each time for 10 minutes.Subsequently, add the permeabilization solution, and incubate for five minutes. Following this, wash the slides with PBS twice, each time for five minutes. In the last wash, add 200 microliters of PBS, containing 0.25%BSA, directly on the slides for five minutes.Incubate the slides with the primary anti-Nanog antibody at 1.25 micrograms per milliliter overnight at 4 degrees Celsius in PBS containing 5%FBS. Wash the slides with PBS twice, each time for 10 minutes. In the last wash, use 200 microliters of PBS containing 0.25%BSA for five minutes.Subsequently, incubate the slides with 100 microliters of RAB-HRP secondary antibody from a ready-to-use kit for 45 minutes. Then, wash the slides with PBS three times, each time for five minutes to remove the secondary antibody. After that, dilute DAB in the substrate buffer, provided by the ready-to-use kit.Vortex vigorously. Add 100 microliters of DAB solution, directly onto each slide, and incubate for a maximum of 10 minutes at room temperature. Next, remove DAB solution by rinsing the slides with water.Counterstain the slides with fast red solution for two minutes. Then, wash the slides with water again, briefly. Following that, dehydrate them with 95%ethanol for five minutes, followed by 100%ethanol twice, each time for five minutes.After that, mount the slides with quick-hardening mounting medium. Observe the slides under the microscope in brightfield at 20x to avoid background. In this procedure, scan the slides and choose the two best quality sections on each slide with the maximum possible distance in-between base on tissue integrity, quality of the staining, and counterstaining.Count the SA-Beta-Gal positive cells in these two sections and calculate the mean value. Then, determine the rank of the pixel size by manually selecting the smallest and the largest positive SA-Beta-Gal cells. In ImageJ Interface, click Analyze, Tools, ROI Manager, Analyze, Set Measurement.Then, select Area. Use the selection tool and surround the smallest and biggest positive SA-Beta-Gal cell, and add it to the ROI Manager by clicking on, Add. After that, measure the sizes, using the Measure button, and save the values for later use.Next, adjust the threshold parameter to ensure all the visible positive cells are selected. Convert the image to grayscale by clicking, Image, Type, then 8-bit. Afterward, go to Image, Adjust, and Threshold.Move the second cursor until all the positive SA-Beta-Gal cells are covered in red, then click, Apply. Analyze the particles by clicking, Analyze, Analyze Particles, followed by Size and apply the value defined earlier. Enter Circularity, 0.00-1.00, and click OK.A summary of all the counted particles will be shown in the ROI Manager.Now, transfer all the selected particles to the original image. Adjust the selection manually to ensure accurate quantification. Add positive cells or remove false positive cells.Finally, measure the area by outlining the section using the selection tool. Click ROI Manager, Add, then Measure. Under the microscope, count the Nanog positive cells in the brightfield manually at 20x magnification.Shown here is a schematic representation to evaluate in vivo reprogramming and senescence level after muscle injury. Here are the representative pictures of SA-Beta-Gal and Nanog staining on the frozen sections of damaged skeletal muscle. These images show SA-Beta-Gal staining, counterstained with eosin, and the staining of Nanog, counterstained with fast red.The non-injured muscles are shown on the top, whereas the injured muscles are shown at the bottom. This graph shows the quantification and correlation of SA-Beta-Gal positive and Nanog positive cells in consecutive sections at 10 days post-injury. While attempting this procedure, it's important to remember to always include a negative control to ensure the specificity of the staining.Following this procedure, all the methods, like immunofluorescence, choosing different marker of senescence, all cell types can be performed in order to answer additional questions, like the cellular identity of the senescent cells. After each development, this technique paved the way for researchers in the field of senescence, or tissue repair and regeneration, to explore how cellular senescence can promote cellular plasticity to facilitate tissue repair and regeneration in mammals. So after watching this video, you should have a good understanding of how to identify both senescent and reprogrammed cells in vivo.

evaluation-injury-induced-senescence-vivo-reprogramming-skeletal

Research

JoVE Journal

Developmental Biology

9.4K Views.  Institut Pasteur. The overall goal of this protocol is to evaluate both in vivo senescence and reprogramming in the skeletal muscle upon tissue injury. This method can help to address critical questions in the regenerative medicine field, such as how to enhance cellular plasticity to promote muscle regeneration. The main advantage of this technique is to provide a reliable method to detect and quantify senescence cells in the resident environment.Cell senescence has been recently implicated in tissue re...