Animal models
Animal experiments were in accordance with the Institutional Animal Care and Use Committee (IACUC, protocols 202100001/2) approved by the New York University Grossman School of Medicine. All mice were on a C57BL/6 J background and acquired from the colony at the National Institute on Aging of the National Institutes of Health. Ages ranged from 3–6 months for young, 23–25 months for aged, and 28–31 months for geriatric mice. All mice were male to accommodate sufficient numbers in aged cohorts, and were maintained in 12 h/12 h light/dark conditions in a controlled environment held at 21.2 oC, and 58% humidity.
Cell culture and staining
C2C12 cells were a gift from Stefano Biressi. Cells were cultured according to ATCC, growth media (C2GM, 10% FBS (Omega Scientific), 1X Penicillin/Streptomycin (Gibco, 15140122), and DMEM (Corning, MT10013CV)). C2C12s were used between passages 5 and 25. To induce senescence, C2C12 cells were plated between 1666–5000 cells/cm2 on day 1. On Day 2, cells were treated with 300 or 500 µM of H2O2 (EMD Millipore, 386790-100 ML), 2 or 10 µM of Etoposide (Millipore Sigma, 34-120-525MG), or 0.08 or 0.4 µM of Doxorubicin (Cayman Chemical, NC1706390) for 2 h, treatment was removed, and fresh C2GM was added to cells that were cultured for an additional 3 days and then fixed with 4% PFA. For ABT-263 treatment, the same protocol was followed, and on Day 5, cells were treated with a dose-response of ABT-263 (TargetMol, T2101) for 24 h before fixing.
3T3-L1 cells were acquired from ATCC and cultured accordingly, growth media (3TGM) contained 10% calf serum (HyClone, SH30072.02) and 1X Penicillin/Streptomycin, in DMEM. 3T3-L1 were used between passages 3 and 25. To induce senescence, 3T3-L1 cells were plated between 833-2000 cells/cm2 on day 1. On Day 2, cells were treated with 150 or 300 µM of H2O2 (EMD Millipore, 386790-100 ML) for 4 h; or 1 or 3 µM of Etoposide, or 0.03 or 0.1 µM of Doxorubicin overnight (16hrs); treatment was removed, and fresh 3TGM was added to cells that were cultured for an additional 2-3 days and then fixed with 4% PFA.
Senescence was detected with the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, 9860S) according to the manufacturer’s protocol, with pH adjusted to 6.0. Brightfield and fluorescent images were captured on the Keyence BZ-X810 microscope. DAPI-stained and overlaid substrate-positive (blue) cells were quantified as senescent.
For immunostaining, cells were fixed with 4% paraformaldehyde (PFA) for 10 min, rinsed in 1X PBS, permeabilized with 0.1% TritonX (Fisher Bioscientific, BP151-100) for 30 min, and blocked with 1% BSA (GeminiBio, 700-109 P)/10% DS (Equitech Bio Inc, NC0629457)/1X PBS/0.1% tween (Fisher Scientific, BP337-100) for 1.5 h. Following this, the cells were incubated with primary antibodies and diluent (1% BSA/10% DS/1X PBS) for 18–20 h. Antibodies included: γH2A.X (Cell Signaling Technology, 9718), Ki67 (Abcam, ab15580), Cleaved-Caspase-3 (Cell Signaling Technology, 9661). After washing with 1X PBS to remove the primary antibodies, cells were incubated with diluent and secondary antibodies for 2 h. Secondary antibodies included: Alexa Fluor 555 Donkey anti-Rabbit (Thermo Scientific, A-31572). Cells were then washed with 1X PBS and counterstained with DAPI (Invitrogen, D1306). All samples were maintained at 4 °C until imaging with the Nikon Eclipse Ti2-LAPP Inverted Microscope System. Images were taken at 20 x with 1 to 5 images taken per well. Images were then quantified with NIS-Elements AR Analysis Software, Nikon Instruments, for positivity of fluorescence and/or intensities.
RT-qPCR
Total RNA extraction from cultured cells was performed according to the manufacturer’s protocol with the RNeasy Micro or Plus Mini Kit (Qiagen, 74004, 74134). RNA quantitation was assessed on the Thermo Scientific NanoDrop One, and cDNA was generated with the RevertAid RT Reverse Transcription Kit (Thermo Scientific, K1691). The mRNA expression was determined on a QuantStudio5 Real-Time PCR System using FastStart SYBR Green Master (Roche, 04673484001). Relative expression levels were calculated with the 2-ΔΔCT method using GAPDH as the reference gene, and each independent experimental replicate included 2-3 technical replicates. Gene expression noted as undetectable by the instrument was assigned a value of 40 Ct. Primer (Thermo Scientific) sequences are in Supplementary Table 1 and were used at 0.2 μM concentration.
Muscle injury
Mice were anesthetized with isoflurane, and injury was induced with 90ul of 1.2% BaCl2 (Sigma-Aldrich, B0750) injected intramuscularly into the lower hindlimb muscles, which were isolated at the days post injury (DPI) noted in the main text. The gastrocnemius was used to isolate cells from injured SkM, and the tibialis anterior (TA) were isolated for histological analysis.
Cell isolation and fluorescence activated cell sorting
To isolate cells from muscle tissue, dissected tissue was minced with scissors and then incubated in 760 U/mL collagenase type 2 (Worthington Biochemical, LS004177) in Ham’s F10 (Cytiva, SH30025.01) for ~1 h in a 37 °C shaking water bath, then washed with wash media (WM, 10% Horse Serum (HS) (Thermo Scientific, 16050122) in Ham’s F-10), and digested again in a 1 to 8.5 dilution of both 1000 U/ml collagenase type 2 (Worthington Biochemical, LS004177) and 11 U/ml dispase (Thermo Scientific, 17105041) in WM for 30 min in a 37 °C shaking water bath. The samples were triturated seven times with a syringe fixed with a 21-gauge needle, diluted in wash media, and then passed through a 40 μm strainer (Falcon, 352340). The single-cell suspension was stained with fluorescent-conjugated antibodies for 30 min at 4 °C. Fluorescence-conjugated antibodies included CD-45 (BioLegend, 103101), Sca-1 (BioLegend, 108120), VCAM (BioLegend, 105720), CD-31 (BioLegend, 102510). Cells were then passed through 35 μm blue-top filters (Falcon, 352235) and sorted using the Sony MA900 Multi-Application Cell Sorter Software (Version 3.3.0) for an enriched fraction of endothelial cells (Enr-ECs), immune cells (ICs), fibroadipogenic progenitors (FAPs), and satellite cells (SCs) (gating schemes in Supplementary Fig. 7H)41,42,43,44. Cells were plated in ECM-coated (Sigma Aldrich, E6909) 96-well plates containing WM, centrifuged at 500 x g for two minutes, and incubated overnight to allow for adherence. After 12 h, cells were fixed with 4% PFA for 10 min and washed with 1X PBS (Gibco, 14-200-075). The cells were then stored at 4 °C until imaging.
For EdU staining, cells were sorted using the Sony MA900, plated on ECM-coated 96-well plates containing growth media ((GM), 10% HS, 20% FBS, 1:100 Penicillin/Streptomycin, and 2.5 ng/ml FGF (PeproTech, 100-18B) in DMEM), then incubated for 3 days (SCs or FAPs) with a pulse of EdU (5 µM) five hours prior to fixing with 4% PFA for 10 min and washed with 1X PBS. EdU incorporation was detected with the Alexa Fluor 555 Click-iT™ Plus EdU Cell Proliferation Kit (Thermo Scientific, C10638) using the manufacturer’s recommended protocol and stored at 4 °C until imaging.
For ABT-263 treatment of FACS isolated FAPs and SCs, 3 DPI muscle tissue from two 5 month old mice were combined and this was done twice to make two input samples. Each sample was sorted using the Sony MA900, plated on ECM-coated 96-well plates containing wash media (WM; 10% HS in Ham’s F-10) at a cellular density of 5000 cells per well. Each sample was divided into four replicates. After incubating for 12 h, cells were treated with a dose-response of ABT-263 (TargetMol, T2101) as noted in Supplementary Fig. 13 for 48 h before fixing.
To isolate chondrocytes from the knee joints, dissected cartilage was collected using a scalpel as previously described, except for minimizing endochondral bone carryover45. The cartilage was then incubated in 760 U/mL collagenase type 2 in Ham’s for about 2 h at 37 °C shaking water bath. The samples were then passed through a 40 μm filter (Falcon, 352340). The single-cell suspension was stained with fluorescent-conjugated antibodies for 15 min at 4 °C. Fluorescent antibodies included Terr-119 (Invitrogen, 48-5921-82) and CD-45 (BioLegend, 103101). Cells were then stained with Propidium Iodide (Invitrogen, P3566), passed through blue-top filters, and sorted using the Sony MA900 Multi-Application Cell Sorter (Supplementary Fig. 14E). Cells were plated on ECM-coated 96-well plates in WM, centrifuged at 500 x g for 2 min, incubated overnight, and fixed 12 h later with 4% PFA for 10 min. All samples were maintained at 4 °C until imaging.
To detect senescence-associated β-galactosidase, the FastCellular Senescence Detection Kit (MP Biomedical, 092690301) was used prior to sorting according to the manufacturer’s protocol. Briefly, samples were incubated at 37 °C in Bafilomycin A1 for 1 h, stained for 30 min at 37 °C with SPiDER-β-gal/Bafilomycin A1, resuspended in WM and maintained at 4 °C until analyzed on the Sony MA900 (Fig. 1C).
Skeletal muscle histology and immunofluorescence
Tibialis anterior (TA) muscles, injured 3 days prior, were isolated on the tibia and fixed in 0.5% paraformaldehyde solution at 4 °C for 5 days. Tissues were then washed with 1X PBS overnight, followed by detachment from the tibia and incubation in 30% sucrose overnight, both at 4 °C. The TA tissues were then suspended in Tissue-Plus O.C.T. Compound (Fischer Scientific, 23-730-57) and frozen in dry ice-cooled isopentane. Cryosections were generated on a Leica cryostat at a thickness of 12 μm, allowed to dry for 30 min, and stored at −80 °C.
For immunocytochemical analysis, cryosections were rehydrated in 1X PBS, permeabilized in 0.1% Triton-X for 20 min, blocked in diluent (1% BSA/10% DS/1X PBS) with 0.1% tween for 2 h, then incubated with primary antibodies in diluent for 18–20 h at 4 °C. Primary antibodies consisted of laminin (Santa Cruz Biotechnology, sc-59854), γH2AX (Cell Signaling Technology, 9718), Ki67 (Abcam, ab15580), CD31 (BD Pharmingen, 553370) and PDGFRα (R&D Systems, AF1062). Cryosections were then washed with 1X PBS and incubated with secondary AlexaFluor-conjugated antibodies in diluent for 2 h. Finally, samples were washed with 1X PBS, counterstained with DAPI, and coverslip mounted with FluoroGel medium (Electron Microscopy Sciences, 16985-10).
Knee joint processing for cartilage histology
Samples were fixed in 10% buffered formalin for 72 h and decalcified in 10% formic acid for 10 days. Decalcified samples were embedded in paraffin, and 4 μm sections were cut perpendicular to the cartilage surface. Sections were stained with Safranin O/Fast green (Electron Microscopy Sciences, 477-73-6 / Sigma-Aldrich, 2353-45-9) using routine methods. Cartilage destruction in the mice was examined using Safranin O staining.
Fluorescence imaging
Fluorescent images were acquired using the Nikon Eclipse Ti2-LAPP Inverted Microscope System equipped with the NIS-Elements Advanced Research acquisition software (Version 5.42.03). Three representative high-magnification images were acquired with a 20X objective per well. For cryosectioned samples, 3–5 representative high-magnification images were acquired with a 60X oil immersion objective. For EdU-stained samples, the entire well was imaged. Z-stacked Images were taken in 1 µm increments. Exposures and look-up tables (LUTs) were held constant for each independent experiment.
Pre-processing pipeline
Image pre-processing was performed with NIS-Elements Advanced Research (AR) Analysis Software (Nikon Instruments, Version 5.42.03). Initial preprocessing started with a 3D deconvolution to remove noise from a Z-stacked image. Then, a max intensity projection was used to allow for all fluorescence to appear on a single plane and ease the analysis of the image. To further refine the images, a low-pass filter was used to pass only details larger than a set pixel value and remove small irregularities. Finally, a rolling ball function was used to further differentiate background and fluorescence intensities.
Nuclei phenotypic extraction
Image analysis was performed with the NIS-Elements AR Analysis Software to achieve single-nuclei resolution. To detect nuclei, a threshold function set for each sample was used on images of wells stained with DAPI, allowing the formation of binary objects in the image. Limitations were made on size and circularity to remove objects such as debris or overlapping nuclei. By creating the binary image, nuclei could be separated, counted, and measured for size, mean intensity, and circularity. To detect foci, a spot detection function was used to count the number of bright circular objects that contrasted from surrounding pixels. Limitations on spots included size, contrast, and intensity. These spots were then aggregated to only count bright spots that were present in the binary data of the detected nuclei. For C2C12 cells/3T3-L1s, as well as primary FAPs and SCs, analyses were automated through the software on whole images. For primary endothelial and immune cells, analyses were done both on whole images and manually to avoid misidentification due to cell aggregation.
UMAP Presentation and clustering
Using R Studio (Version 4.3.1), the four phenotypic measures of nuclei were imported and normalized using a z-score within their respective parameters. To cluster visually similar cells, package factoextra (Version 1.0.7) was used, specifically the function fviz_cluster, which applies a k-means algorithm on the 4 normalized phenotypic measures. Using the R studio package UMAP (Version 0.2.10.0), a two-dimensional visualization (n_neighbors = 20, n_components = 2) of the four parameters was made to localize visually similar cells. To form the senescence score, the prior step was repeated with n_components = 1 and required a high number of cells to achieve optimal dynamic range. For three-dimensional visualization, the prior step was repeated with n_components = 3. The clustering data, which is done at the higher dimension, is then visualized on the lower- dimensional UMAP. Within each UMAP, conditions are equally represented by inputting an equal number of cells from each condition into the platform, ensuring accurate quantification. Data was exported and graphed with GraphPad Prism. See Supplementary Fig. 16 for a diagrammatic overview of these processes.
Statistics and reproducibility
All reported measurements were obtained from distinct experimental samples. Experiments were performed at least three times, using a single biological/experimental (not technical) replicate per condition in each experiment. “n” numbers indicate independent experiments unless otherwise noted. Statistical analyses were performed using GraphPad Prism (version 9.5.0). Error bars represent mean ± standard error of the mean (SEM). Statistical tests were conducted using two-sided t tests unless otherwise specified. Significance is indicated as follows: p > 0.05 (n.s. – not significant); p ≤ 0.05 (*); p ≤ 0.01 (**); p ≤ 0.001 (***); p ≤ 0.0001 (****). No data were excluded from analysis except for one replicate of RT-qPCR due to a technical issue. No statistical method was used to predetermine sample size. Experiments were randomized, and investigators were not blinded to sample allocation or condition during experiments or during immunofluorescence image analysis.
Figure 1B includes n = 3 experimental replicates, with per-condition cell counts as follows: X-Gal, 100-300 cells; Ki-67, 100-300 cells; γH2A.X, 150–250 cells; and cleaved caspase, 100-600 cells; Fig. 1C includes n = 3 experimental replicates, with 400-4000 SA-β-Gal cells per condition; Fig. 1D includes n = 4 experimental replicates, each with two to three technical replicates; Fig. 1E includes n = 3 experimental replicates, with 4000–8000 cells per replicate; Fig. 3E, F includes n = 3 experimental replicates, with 1000–1500 cells per replicate; Fig. 4B–E includes n = 3 biological replicates, with cell counts per replicate as follows: FAPs, 1.5-5.0 × 10³ cells; SCs, 1.0-1.2 × 10³ cells; Fig. 5B, C include n = 3 biological replicates, with 150-200 cells per condition; Fig. 5E, F include n = 3 biological replicates, with 300-600 cells per condition; Supplementary Fig. 4 includes n = 3 experimental replicates per inducer with X-Gal n = 100-600 cells, γH2A n = 100-500 cells, and Ki-67 = 200–350 cells per condition; Supplementary Fig. 4 includes n = 3 experimental replicates per inducer with n = 800–1100 cells per condition with γH2A n = 250–600 cells, and Ki-67 n = 150–600 cells per condition; Supplementary Fig. 7A–F includes n = 3 biological replicates, with cell counts per replicate as follows: FAPs, 200 cells; SCs, 200 cells; Supplementary Figs. 9C, 5C includes n = 3 biological replicates, with 100-300 per condition; Supplementary Figs. 9F, 5F includes n = 3 biological replicates, with 200-600 cells condition; Supplementary Fig. 12B includes n = 4 biological replicates, with 1000–2000 cells per condition; Supplementary Fig. 12C includes n = 4 biological replicates, with 500–1500 cells per condition; Supplementary Fig. 13C, n = 8 experimental replicates with cell input pulled from 2 mice to make 4 replicates. This was done twice; Supplementary Fig. 13E includes n = 7 experimental replicates with cell input pulled from 2 mice to make 4 replicates, and 2 mice to make 3 replicates. Supplementary Fig. 14C, D includes n = 5 biological replicates, with n = 82–96 cells per condition, n = 123–134 cells per condition, for chondrocytes and immune cells, respectively.
Figure construction
Figures were assembled using BioRender, and schematics were created in BioRender where noted in the figure legends. Graphs were made using GraphPad Prism (Version 9.5.0) unless otherwise stated. Figure 2A was made using the Rstudio package ggplot2 (version 3.3.4) and extension ggally (Version 2.2.1). Figure 2C was made using the Rstudio package kohonen: supervised and unsupervised self-organizing maps (Version 3.0.12). Three-dimensional UMAPs were made using the Rstudio package plotly (Version 4.10.4). FACS plots were constructed using FlowJo (Version 9).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
