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 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 β-galactosidase (SA-β-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 β-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 (IHC). The major limitation of the SA-β-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-β-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use 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.
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 (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-β-Gal) and reprogramming (via IHC staining of Nanog).
Senescence-associated beta-galactosidase (SA-β-Gal) assay is the most commonly used assay to detect senescent cells both in culture and in vivo15. Compared to other assays, the SA-β-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-β-Gal assay with other markers using IHC. However, the SA-β-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-β-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.
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
2. SA-β-Gal Staining on Frozen TA Muscle Section
3. Immunohistochemistry Using Anti-Nanog Antibody
4. Analysis and Quantification
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 dramatically reduced while remaining visible in certain regions. 10 DPI is a good time point to detect senescent cells by SA-β-Gal, since there are fewer necrotic and inflammatory cells present in the muscle to interfere with the staining. To determine the specificity of the staining, TA injected with PBS from the same mouse (Figure 1A) is used as a critical negative control.
To ensure a better and more precise evaluation of the SA-β-Gal positive cells, sections from different planes of the TA muscle are placed in the same slide (Figure 1B). Counter staining with eosin is important for the automatic quantification of the SA-β-Gal positive cells by ImageJ software. Eosin counter staining outlines the section, which allows the digital scanner to detect the sections with the correct focus. It is important to carefully define the range and the threshold of the detection (Figure 2A-D). In addition, a manually curated process is essential to permit more accurate detection and quantification (Figure 2E).
Cellular senescence facilitates in vivo reprogramming in muscle
Reprogrammable mouse model (i4F) provides an ideal system to evaluate the impact of senescence on cellular plasticity and regeneration. Upon muscle injury, i4F mice are treated with dox to induce reprogramming in vivo. 7 days dox (1 mg/mL) treatment is sufficient to induce reprogramming on the cellular level, while still being well tolerated by mice (Figure 3A). Therefore, we harvest injured muscles at 10 DPIs from i4F mice treated with dox for 7 days.
Although it is possible to perform co-staining of SA-β-Gal with Nanog, it is not recommended for quantification due to potential interfering staining. As mentioned above, counter staining is essential for digital scanner detection. The best counter staining for the SA-β-Gal together with Nanog is fast red orhematoxylin. However, counter staining might mask over either the SA-β-Gal or Nanog signal. Therefore, for more accurate quantification, it is better to perform them separately on consecutive slides (Figure 3B). By quantifying SA-β-Gal positive and Nanog positive cells from adjacent slides, we established a positive correlation between them (Figure 3C), suggesting a potential involvement of senescence on cellular plasticity and regeneration.
Figure 1: Evaluate senescence level after muscle injury. (A) Schematic representation of the muscle injury strategy used to induce senescence. (B) Schematic representation of the muscle section preparation. (C) Representative images of SAβGal staining counterstained with eosin. Arrows point to the SA-β-Gal+ cells. Scale bars = 50 µm. (D) Quantification of SA-β-Gal+ cells in injured and non-injured TA-muscle. Each dot corresponds to an individual animal. Statistical significance was assessed by the two-tailed Student´s t-test: ***p <0.001. Please click here to view a larger version of this figure.
Figure 2: Quantification of SA-β-Gal+ cells by ImageJ software. (A-D) Screen shots of a muscle section in the ImageJ software interface. Screen shot of converting a muscle section image to gray scale (A); Selecting all the SA-β-Gal+ cells in the section (B); Analyzing the selected particles (C); Summary of all the counted particals in the ROI manager (D). (E) Screen shot of the manual curation process. Please click here to view a larger version of this figure.
Figure 3: Evaluation of in vivo reprogramming after muscle injury. (A) Schematic representation to evaluate in vivo reprogramming and senescence level after muscle injury. (B) Representative images of SA-β-Gal and Nanog staining on frozen sections of damaged skeletal muscle. SAβGal staining counterstained with eosin (left); immunohistochemical staining of Nanog counterstained with fast red (right). Non-injured muscles are shown on the top and injured muscle below. Scale bars = 50 µm. (C) Quantification and correlation of SA-β-Gal + and Nanog+ cells in consecutive sections (n = 9 mice, value represents the average of 2 sections per mouse). Please click here to view a larger version of this figure.
Volume for 50 mL | |
100 mM stock K3Fe(CN)6 solution | 2 mL |
100 mM stock K4Fe(CN)6 solution | 2 mL |
1 M MgCl2 | 100 μL |
50 mg/mL X-Gal | 400 μL |
PBS (pH 5.5) | 45.50 mL |
Table 1: Composition of 50 mL X-gal solution.
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 β-galactosidase (SA-β-Gal) assay is used to detect in vivo senescent cells in the skeletal muscle. This assay detects the increased lysosomal β-galactosidase activity at suboptimum pH (6.0 or 5.5), associated specifically with senescent cells, while this enzyme's activity is typically measured at acidic pH 4.516,17. Therefore, it is important to adjust the pH (pH = 6 or 5.5) to ensure a specific detection of senescence-associated activity. In addition, counter staining with eosin is essential for the automatic quantification of SA-β-Gal+ cells, where the weak and diffuse signals are not counted. To avoid potential variability, it is preferable that the same person performs the entire counting procedure.
Although the SA-β-Gal assay is the most widely used and accepted biomarker for senescent cells, it is not an exclusive marker for senescence. It has been suggested that over-confluent cells in culture might cause false positivity for SA-β-Gal18. The sensitivity of the assay can be cell type and tissue type dependent in vivo19. Therefore, it is necessary to use other independent canonical markers, such as lack of proliferation, increased expression of senescence mediators (p16, ARF, p53, p21, and p27), and the secretion of various SASP factors, to further confirm and characterize senescence in vivo. Moreover, proper negative controls are indispensable for interpreting results, especially for in vivo study.
Despite the fact that SA-β-Gal assay is not perfect, it does provide particularly valuable information for in vivo study. It permits the detection of senescent cells in their resident environment with intact tissue architecture, providing critical information facilitating the further understanding of the senescent cells' role in different physiological and pathological contexts. Moreover, it can be coupled with the immunostaining of other markers, such as cell surface markers to determine the cellular identity of senescent cells; or stemness markers to examine the potential involvement of senescence in regeneration and tumorigenesis. Previously, we performed the SA-β-Gal assay with immunohistochemical staining of Nanog in the same section or in close proximity to investigate the potential link between senescence and in vivo reprogramming8. While this protocol is focused on skeletal muscle, it can be certainly extended to other tissues.
Recently, cellular senescence has been implicated in tissue repair and regeneration, most likely via SASPs7,8,9. Understanding the mechanisms of how senescence contributes to tissue repair and regeneration will certainly have a tremendous impact on regenerative medicine. This assay provides an important and valuable tool to facilitate the identification and quantification of senescent cells in vivo.
The authors have nothing to disclose.
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'Excellence Revive, Investissement d'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.
K3Fe(CN)6 | Sigma | 13746-66-2 | For SA-β Gal staining solution |
K4Fe(CN)6 | Sigma | 14459-95-1 | For SA-β Gal staining solution |
MgCl2 | Sigma | 7786-30-3 | For SA-β Gal staining solution |
X-Gal | Sigma | B4252 | For SA-β Gal staining solution |
Doxycycline | Sigma | D3447 | For inducing in vivo reprogramming |
Cardiotoxin | Lotaxan Valence, France | L8102 | For muscle injury |
Glutaraldehyde | Sigma | 111-30-8 | For Fixation solution |
Paraformaldehyde | Electron microscopy science | 50-980-487 | For Fixation solution |
NaCitrate : Sodium Citrate monobasic bioxtra, anhydre | Sigma | 18996-35-5 | For permeabilization solution |
Triton | Sigma | 93443 | For permeabilization solution |
Bovine Serum Albumin | Sigma | A3608 | Washing solution |
Antibody anti- Nanog | Cell signalling | 8822S | Rabbit monoclonal antibody |
EnVision+ Kits (HRP. Rabbit. DAB+) | Dako | K4010 | For Nanog revelation |
Eosin 1% | Leica | 380159EOF | Counterstainning |
Fast red | Vector Laboratories | H-3403 | Counterstainning |
Thermo Scientific Shandon Immu-Mount | Fisher scientific | 9990402 | Mounting solution |
Quick-hardening mounting medium for microscopy : Eukitt® | Sigma | 25608-33-7 | Mounting solution |
Microscope Phase Contrast Brightfield CKX41: 10X-20X-40X objectives | Olympus | CKX41 | Microscope for Nanog quantification |
Mouse: i4F-A | Abad et al., 2013 | N/A | Reprogrammable mouse model |
Skeletal muscle, Tibialis Anterior | |||
Slide Scanner | Zeiss | Axio Scan Z1 | slides scanning |