1. Stem Cells and Regenerative Medicine

The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation

  1. Erik Jacques
  2. Yinni Kuang
  3. Allison P Kann
  4. Fabien Le Grand
  5. Robert S Krauss
  6. Penney M Gilbert  Is a corresponding author
  1. Institute of Biomedical Engineering, University of Toronto, Canada
  2. Donnelly Centre, University of Toronto, Canada
  3. Department of Cell and Systems Biology, University of Toronto, Canada
  4. Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at Mount Sinai, United States
  5. Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, United States
  6. Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, United States
  7. Université Claude Bernard Lyon 1, CNRS UMR 5261, INSERM U1315, Institut NeuroMyoGène - Pathophysiology and Genetics of Neuron and Muscle, France
https://doi.org/10.7554/eLife.81738
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Cite this article
  1. Erik Jacques
  2. Yinni Kuang
  3. Allison P Kann
  4. Fabien Le Grand
  5. Robert S Krauss
  6. Penney M Gilbert
(2022)
The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation
eLife 11:e81738.
https://doi.org/10.7554/eLife.81738
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9 figures, 3 tables and 1 additional file

Figures

Figure 1 with 2 supplements
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A three-dimensional (3D) murine skeletal muscle myotube template with a 96-well footprint.

(A) Schematic overview of the strategy used to generate myotube templates with an associated timeline for downstream culture (made with BioRender). (B) Representative confocal stitched images of …

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Figure 1—figure supplement 1
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Optimization of cell seeding density.

(A) Representative confocal stitched images of myotube templates labelled for sarcomeric α-actinin (SAA; magenta) at day 5 using 10,000, 25,000, or 50,000 myoblasts per tissue. Scale bar, 1 mm. (B) …

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Figure 1—figure supplement 2
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Accelerated maturity and prolonged culture of myotube templates over two-dimensional (2D) monolayers.

(A) Low- and (B) high-magnification representative confocal images of myotube templates labelled for sarcomeric α-actinin (SAA) (magenta) at days 5, 8 and 12 of differentiation in a 3D gel (top row) …

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Figure 2 with 1 supplement
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Engrafted muscle stem cells (MuSCs) persist in myotube template cultures and achieve a steady-state population density.

(A) Schematic overview of the engraftment of freshly isolated MuSCs and the timeline for downstream analysis (made with BioRender). (B) Representative confocal images of myotube templates …

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Figure 2—figure supplement 1
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Population purity in magnetic-activated cell sorting (MACS) isolated muscle stem cells (MuSCs).

(A) Flow cytometry gating of MuSCs enriched from the hindlimb muscles of a Pax7-nGFP mouse using the MACS-based workflow. (B) Representative confocal image of MuSCs (DAPI: cyan, YFP: yellow, Pax7: …

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Figure 3 with 3 supplements
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Muscle stem cells (MuSCs) engrafted within engineered muscle tissue exit cell cycle and inactivate.

(A) Representative confocal image of a mononuclear cell (DAPI: cyan) positive for YFP (yellow), caveolin-1 (magenta), and c-FOS (white) at 1 day post-engraftment (DPE) (top), and a c-FOS- cell at 7 …

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Figure 3—figure supplement 1
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Persistent CalcR+ population amongst Pax7+ donor cell population at 7 days post-engraftment (DPE).

(A) Representative confocal images of a Pax7+ (magenta) donor cells (yellow) at 1 DPE (top) and 7 DPE (middle and bottom) immunostained for calcitonin receptor (CalcR: white) and counterstained with …

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Figure 3—figure supplement 2
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Characterization of barium chloride-induced injury.

(A) Quantification of sarcomeric α-actinin (SAA) coverage on day 12 of differentiation, and 2 days (day 14) after a physiological salt solution (PSS; carrier control) or 2.4% BaCl2 exposure. n=7–9 …

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Figure 3—figure supplement 3
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Regulation of muscle stem cell (MuSC) pool size in myotube template cultures.

(A) Representative confocal image of YFP+ (yellow) mononucleate donor cells at 7 days post-engraftment (DPE) co-labelled for EdU (red), Ki67 (white), and nuclei (DAPI: cyan), to visualize EdU+Ki67- …

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Figure 4 with 1 supplement
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Permissive culture conditions for a persistent muscle stem cell (MuSC) population in vitro.

(A) Key for figure icons. (B–F) Line graphs of mononucleated DAPI+YFP+Pax7+ cell density at 1, 3, and 7 days post-engraftment (DPE) (left) and pie charts showing the proportion of Ki67 ± cells at 7 …

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Figure 4—figure supplement 1
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Increased YFP coverage when muscle stem cells (MuSCs) engrafted on day 0 of myotube template differentiation.

(A) Representative confocal images of tissues engrafted with 500 MuSCs at day 0 or day 5 of myotube template differentiation, fixed at 7 days post-engraftment (DPE) (i.e. days 7 and 12 of …

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Figure 5 with 5 supplements
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Morphological evolution of engrafted muscle stem cells (MuSCs).

(A) Representative confocal images of MuSCs (DAPI: cyan, YFP: yellow, Pax7: white) with distinct morphological features at 1, 3, and 7 days post-engraftment (DPE). Insets highlight nuclear …

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Pax7+ donor cell morphologies in two (2D) and three dimensions (3D).

(A) Representative confocal images of mononucleated donor cells (YFP:yellow; Pax7:white) after 7 days in a 2D Geltrex-coated Petri dish (left), in 2D co-culture with a myotube monolayer established …

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Figure 5—figure supplement 2
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CellProfiler pipeline for muscle stem cell (MuSC) identification and characterization.

(A) Representative image of the pipeline loaded in CellProfiler. (B) Representative confocal image of mononucleated (DAPI:cyan) donor (YFP:yellow) MuSCs (Pax7:white) at 7 days post-engraftment (DPE) …

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Figure 5—figure supplement 3
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Morphological characterization of Pax7+MyoD and Pax7+MyoD+ muscle stem cells (MuSCs).

(A) Dot plot graphs showing individual Pax7+ donor cells and their associated max/min Feret diameter ratio and nuclear eccentricity at 1 (left), 3 (middle), and 7 days post-engraftment (DPE) (right) …

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Figure 5—figure supplement 4
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ROCK inhibition hastens muscle stem cell (MuSC) inactivation.

(A) Representative confocal images of MuSCs (DAPI: cyan, YFP: yellow, Pax7: white) at 1, 3, and 7 days post-engraftment (DPE) treated with a vehicle control or the ROCK inhibitor Y-27632. Scale bar, …

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Figure 5—figure supplement 5
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ROCK inhibition confers acquisition of quiescent-like morphologies to the Pax7+MyoD+ population.

(A) Distribution plots of individual muscle stem cell (MuSC) max/min Feret diameter bins at 1 (top), 3 (middle), and 7 days post-engraftment (DPE) (bottom) in the control and Y-27632 treatment …

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Figure 6 with 2 supplements
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Engrafted muscle stem cells (MuSCs) display quiescence and niche-related hallmarks.

(A) Representative confocal image of a mononuclear donor cell (DAPI: cyan, YFP: yellow) with neighbouring myotubes (Phalloidin: magenta) and N-cadherin (white) localized to the tip of the donor cell …

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Polarized localization of M-cadherin in mononuclear donor cells at 7 days post-engraftment (DPE).

(A) Representative confocal images of two (Cell #1, left; Cell #2, right) mononucleated donor cells (DAPI: cyan; YFP: yellow) with M-cadherin (M-cad: white, white arrows) labelling restricted to the …

Figure 6—figure supplement 2
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Donor cell polarization of niche markers at 7 days post-engraftment (DPE).

(A) Representative confocal image of a mononucleated donor cell (DAPI: cyan; YFP: yellow) with M-cadherin (white) immunolabelling restricted to the apical side and laminin α-2 (magenta) localized to …

Figure 7 with 2 supplements
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Aberrant pool size maintenance and inactivation in aged muscle stem cells (MuSCs) is rescued by wortmannin.

(A) Quantification of mononuclear DAPI+Centrin 2-GFP (C2-GFP)+Pax7+ cell density per mm2 at 1, 3, and 7 days post-engraftment (DPE) between engrafted young and aged MuSCs ± wortmannin (wort) …

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Wortmannin treatment blunts cell proliferation and increases FoxO3a nuclear localization in young and aged muscle stem cells (MuSCs) cultured in vitro.

(A) Bar graph showing the mean number of MuSCs per mm2 of 2D microwells on days 1 and 3 across experimental conditions (young: dark grey; young + wortmannin: light grey; aged: red; aged + …

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Figure 7—figure supplement 2
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Wortmannin treatment diminishes GFP coverage at 7 days post-engraftment (DPE).

(A) Representative confocal images of the Centrin 2-GFP area coverage (yellow) at 7 DPE from young and aged engrafted muscle stem cells (MuSCs) treated with dimethyl sulfoxide (DMSO) control or …

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Tables

Table 1
Cell culture media and solutions.
MediaComposition
FACS BufferPBS, 2.5% Goat serum (Gibco, #16210072), 2 mM EDTA (Sigma-Aldrich, #E5134)
RBC Lysis BufferddH2O, 0.155 M NH4Cl (Sigma-Aldrich, #A9434), 0.01  M KHCO3 (Sigma-Aldrich, #237205), 0.1  mM EDTA
MACS BufferPBS, 0.5% Bovine serum albumin (BioShop, #9048-46-8), 2 mM EDTA
SAT10DMEM/F12 (Gibco, #11320-033), 1% Penicillin-streptomycin (Gibco, #15140-122), 20% Fetal bovine serum (Gibco, 12483-020), 10% Horse serum (Gibco, #16050-122), 1% Glutamax (Gibco, #35050-061), 1% Insulin-transferrin-selenium (Gibco, #41400-045), 1% Non-essential amino acids (Gibco, #11140-050), 1% Sodium pyruvate (Gibco, #11360-070), 50 µM β-mercaptoethanol (Gibco, #21985-023), 5 ng/mL bFGF (ImmunoTools, #11343625)
Growth media (GM)SAT10 – bFGF, 1.5 mg/mL Aminocaproic acid (Sigma-Aldrich, #A2504)
Differentiation media (DM)DMEM (Gibco, #11995-065), 2% Horse serum, 2 mg/mL Aminocarpoic acid, 10 µg/mL Insulin (Sigma, #I6634), 1% Penicillin-streptomycin
Blocking SolutionPBS, 10% Goat serum, 0.3% Triton X-100 (BioShop, #TRX777)
Physiological Salt Solution (PSS)140 mM NaCl (Sigma-Aldrich, #S5886), 5 mM KCl (Sigma-Aldrich, #P3911), 1 mM MgCl2 (Alfa Aesar, #7786-30-3), 10 mM HEPES (BioShop, #7365-45-9), 10 mM Glucose (Sigma-Aldrich, #G8270), 2 mM CaCl2 (Sigma-Aldrich, #C1016), corrected to pH 7.3–7.4
Wash Media89% DMEM, 10% Fetal bovine serum, 1% Penicillin-streptomycin
Table 2
Antibodies.
AntibodyHost SpeciesDilutionManufacturer
DAPI–1:1000Roche, #10236276001
Phalloidin 568–1:400Life Technologies, #A12380
Propidium iodide–1:1000Sigma-Aldrich, #P4863
DRAQ5–1:800Cell Signaling Technology, #4084L
Anti-sarcomeric α-actininMouse1:800Sigma-Aldrich, #A7811
Anti-GFPChicken1:500Abcam, #ab13970
Anti-Pax7Mouse IgG11.5:1In-house supernatant from hybridoma cell line (DSHB)
Anti-caveolin-1Rabbit1:300Abcam, #ab2910
Anti-c-FOSMouse IgG11:250Santa-Cruz, #sc-166940
Anti-Ki67Rabbit1:300Abcam, #ab16667
Anti-N-cadherinMouse IgG11:250Santa-Cruz, #sc-8424
Anti-MyoDMouse IgG2b1:300Santa-Cruz, #sc-377460
Anti-CalcRRabbit1:250Abcam, #ab11042
Anti-DDX6Rabbit1:400Cederlane Labs, #A300-461A-T
Anti-Integrin α-7Rabbit1:250Abcam, #ab203254
Anti-M-cadherinMouse IgG11:250Santa-Cruz, #sc-81471
Anti-Laminin α-2Rat1:400Abcam, #ab11576
Anti-FoxO3aMouse IgG11:250Santa-Cruz, #sc-48348
Alexa Fluor 488 Anti-mouse IgG (H+L)Goat1:500Invitrogen, #A11001
Alexa Fluor 488 Anti-chicken IgGY (H+L)Goat1:500Invitrogen, #A11039
Alexa Fluor 546 Anti-mouse IgG (H+L)Goat1:250Invitrogen, #A11003
Alexa Fluor 546 Anti-rabbit IgG (H+L)Goat1:250Invitrogen, #A11010
Alexa Fluor 546 Anti-rat IgG (H+L)Goat1:400Invitrogen, #A11081
Alexa Fluor 546 Anti-mouse IgG2bGoat1:300Invitrogen, #A21141
Alexa Fluor 555 picolyl azide–1.2:500Invitrogen, #C10638B
Alexa Fluor 647 Anti-mouse IgG1Goat1:250Invitrogen, #A21240
Alexa Fluor 647 Anti-rabbit IgG (H+L)Goat1:250Life Technologies, #A21245
Table 3
Experimental replicate breakdown and statistical analysis.
FigureIndependent technical and biological replicates (n, N)Images per technical replicate (tissue)n to calculate statistics/error barsStatistical test
1DSAA coverage Day 2: n=12 across N=4 Day 5: n=12 across N=4 Day 10: n=15 across N=5 Day 14: n=15 across N=5 Day 16: n=12 across N=4 Day 18: n=12 across N=4 Fusion index Day 2: n=9 across N=3 Day 5: n=12 across N=4 Day 10: n=18 across N=6 Day 14: n=15 across N=5 Day 16: n=6 across N=2 Day 18: n=12 across N=4SAA coverage: 21 images stitched together Fusion Index: 9SAA coverage Day 2: n=12 Day 5: n=12 Day 10: n=15 Day 14: n=15 Day 16: n=12 Day 18: n=12 Fusion index Day 2: n=9 Day 5: n=12 Day 10: n=18 Day 14: n=15 Day 16: n=6 Day 18: n=12One-way ANOVA with Tukey’s post-test
1EDay 2: n=12 across N=4 Day 5: n=12 across N=4 Day 10: n=9 across N=3 Day 14: n=12 across N=4 Day 16: n=12 across N=4 Day 18: n=9 across N=33 readsDay 2: n=12 Day 5: n=12 Day 10: n=9 Day 14: n=12 Day 16: n=12 Day 18: n=9One-way ANOVA with Tukey’s post-test
2D200 MuSCs 1DPE: n=11 across N=4 3DPE: n=12 across N=4 7DPE: n=12 across N=4 500 MuSCs 1DPE: n=8 across N=3 3DPE: n=8 across N=3 7DPE: n=9 across N=3 1500 MuSCs 1DPE: n=7 across N=3 3DPE: n=9 across N=3 7DPE: n=11 across N=4 2500 MuSCs 1DPE: n=9 across N=3 3PE: n=8 across N=3 7DPE: n=9 across N=325200 MuSCs 1DPE: n=11 3DPE: n=12 7DPE: n=12 500 MuSCs 1DPE: n=8 3DPE: n=8 7DPE: n=9 1500 MuSCs 1DPE: n=7 3DPE: n=9 7DPE: n=11 2500 MuSCs 1DPE: n=9 3PE: n=8 7DPE: n=9One-way ANOVA with Dunnet’s test for each individual timepoint comparing against the 500 MuSC condition
3B1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3251DPE: n=9 3DPE: n=9 7DPE: n=9One-way ANOVA with Tukey's post-test comparing the FOS- proportions of each timepoint
3C1DPE: n=10 across N=3 3DPE: n=11 across N=4 7DPE: n=11 across N=4251DPE: n=10 3DPE: n=11 7DPE: n=11One-way ANOVA with Tukey’s post-test comparing the Ki67- proportions of each timepoint
3En=15 across N=525n=15–
3GPSS n=16 across N=5 2.4% BaCl n=18 across N=625PSS n=16 2.4% BaCl n=18Unpaired t-test of the Ki67- proportions of both conditions
4B1DPE: n=9 across N=3 3DPE: n=10 across N=3 7DPE: n=9 across N=3251DPE: n=9 3DPE: n=10 7DPE: n=9–
4C1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3251DPE: n=9 3DPE: n=9 7DPE: n=9–
4D1DPE: n=6 across N=2 3DPE: n=6 across N=2 7DPE: n=6 across N=2251DPE: n=6 3DPE: n=6 7DPE: n=6–
4E1DPE: n=6 across N=2 3DPE: n=6 across N=2 7DPE: n=6 across N=2251DPE: n=6 3DPE: n=6 7DPE: n=6–
4F1DPE: n=15 across N=4 3DPE: n=14 across N=4 7DPE: n=13 across N=4251DPE: n=15 3DPE: n=14 7DPE: n=13–
5C1DPE: n=916 across N=4 3DPE: n=980 across N=4 7DPE: n=737 across N=325––
5E1DPE: n=639 across N=3 3DPE: n=770 across N=3 7DPE: n=676 across N=3251DPE: n=639 3DPE: n=770 7DPE: n=676One-way ANOVA with Tukey’s post-test
5Fn=676 across N=325Bin 2=147 Bin 3=135 Bin 4=89 Bin 5=69 Bin 6=66 Bin 7=48 Bin 8=44 Bin 9=30 Bin 9+=48One-way ANOVA with test for linear trend
6Cn=8 across N=325––
6Dn=8 across N=345––
7AYoung 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3 Young + wortmannin 1DPE: n=6 across N=2 3DPE: n=6 across N=2 7DPE: n=6 across N=2 Aged 1DPE: n=9 across N=3 3DPE: n=8 across N=3 7DPE: n=9 across N=3 Aged + wortmannin 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=7 across N=325Young 1DPE: n=9 3DPE: n=9 7DPE: n=9 Young + wortmannin 1DPE: n=6 3DPE: n=6 7DPE: n=6 Aged 1DPE: n=9 3DPE: n=8 7DPE: n=9 Aged + wortmannin 1DPE: n=9 3DPE: n=9 7DPE: n=7One-way ANOVA with Dunnet’s test for each individual timepoint comparing against the Young condition
7CYoung 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3 Young + wortmannin 1DPE: n=6 across N=2 3DPE: n=6 across N=2 7DPE: n=6 across N=2 Aged 1DPE: n=9 across N=3 3DPE: n=8 across N=3 7DPE: n=9 across N=3 Aged + wortmannin 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=7 across N=325Young 1DPE: n=9 3DPE: n=9 7DPE: n=9 Young + wortmannin 1DPE: n=6 3DPE: n=6 7DPE: n=6 Aged 1DPE: n=9 3DPE: n=8 7DPE: n=9 Aged + wortmannin 1DPE: n=9 3DPE: n=9 7DPE: n=7One-way ANOVA with Tukey’s post-test comparing the conditions against each other at the 3 DPE timepoint
7DYoung 1DPE: n=6 across N=2 3DPE: n=6 across N=2 7DPE: n=5 across N=2 Young + wortmannin 1DPE: n=5 across N=2 3DPE: n=5 across N=2 7DPE: n=5 across N=2 Aged 1DPE: n=5 across N=2 3DPE: n=8 across N=3 7DPE: n=10 across N=3 Aged + wortmannin 1DPE: n=5 across N=2 3DPE: n=6 across N=2 7DPE: n=6 across N=225Young 1DPE: n=6 3DPE: n=6 7DPE: n=5 Young + wortmannin 1DPE: n=5 3DPE: n=5 7DPE: n=5 Aged 1DPE: n=5 3DPE: n=8 7DPE: n=10 Aged + wortmannin 1DPE: n=5 3DPE: n=6 7DPE: n=6One-way ANOVA with Tukey’s post-test comparing the conditions against each other at the 3 DPE timepoint
7EYoung n=9 across N=3 Young + wortmannin n=6 across N=2 Aged n=9 across N=3 Aged + wortmannin n=9 across N=325––
7FYoung n=9 across N=3 Young + wortmannin n=6 across N=2 Aged n=9 across N=3 Aged + wortmannin n=9 across N=325Young n=9 Young + wortmannin n=6 Aged n=9 Aged + wortmannin n=9One-way ANOVA with Tukey’s post-test
7GYoung n=9 across N=3 Young + wortmannin n=6 across N=2 Aged n=9 across N=3 Aged + wortmannin n=9 across N=325Young n=9 Young + wortmannin n=6 Aged n=9 Aged + wortmannin n=9One-way ANOVA with Tukey’s post-test
F1-S1B10,000 n=12 across N=4 25,000 n=12 across N=4 50,000 n=12 across N=421 images stitched together10,000 n=12 25,000 n=12 50,000 n=12One-way ANOVA with Tukey’s post-test
F1-S2C3D 1DPE: n=6 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3 2 D 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=321 images stitched together3D 1DPE: n=6 3DPE: n=9 7DPE: n=9 2D 1DPE: n=9 3DPE: n=9 7DPE: n=9One-way ANOVA with Tukey’s post-test
F1-S2D3D 1DPE: n=6 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3 2 D 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3253D 1DPE: n=6 3DPE: n=9 7DPE: n=9 2D 1DPE: n=9 3DPE: n=9 7DPE: n=9One-way ANOVA with Tukey’s post-test
F1-S2E3D 1DPE: n=6 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3 2 D 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=323D 1DPE: n=6 3DPE: n=9 7DPE: n=9 2D 1DPE: n=9 3DPE: n=9 7DPE: n=9One-way ANOVA with Tukey’s post-test
F1-S2F3D 1DPE: n=6 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3 2 D 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=323D 1DPE: n=6 3DPE: n=9 7DPE: n=9 2D 1DPE: n=9 3DPE: n=9 7DPE: n=9One-way ANOVA with Tukey’s post-test
F1-S2G3D 1DPE: n=6 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3 2 D 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=323D 1DPE: n=6 3DPE: n=9 7DPE: n=9 2D 1DPE: n=9 3DPE: n=9 7DPE: n=9One-way ANOVA with Tukey’s post-test
F2-S1Cn=11 across N=425n=11–
F3-S1B1DPE: n=8 across N=3 3DPE: n=7 across N=3 7DPE: n=15 across N=5251DPE: n=8 3DPE: n=7 7DPE: n=15One-way ANOVA with Tukey’s post-test
F3-S2ABI n=8 across N=3 PSS n=7 across N=3 2.4% BaCl n=9 across N=321 images stitched togetherBI n=8 PSS n=7 2.4% BaCl n=9One-way ANOVA with Tukey’s post-test
F3-S2BBI n=7 across N=3 PSS n=9 across N=3 2.4% BaCl n=9 across N=325BI n=7 PSS n=9 2.4% BaCl n=9One-way ANOVA with Tukey’s post-test
F3-S3C200 MuSCs n=11 across N=4 500 MuSCs n=15 across N=5 1500 MuSCs n=16 across N=5 2500 MuSCs n=13 across N=425200 MuSCs n=11 500 MuSCs n=15 1500 MuSCs n=16 2500 MuSCs n=13One-way ANOVA with Tukey’s post-test
F3-S3D200 MuSCs n=12 across N=4 500 MuSCs n=9 across N=3 1500 MuSCs n=12 across N=4 2500 MuSCs n=9 across N=425200 MuSCs n=12 500 MuSCs n=9 1500 MuSCs n=12 2500 MuSCs n=9One-way ANOVA with Tukey’s post-test
F4-S1BDay 5 n=15 across N=5 Day 0 n=11 across N=425Day 5 n=15 5 Day 0 n=11Unpaired t-test
F5-S1B2D n=9 across N=3 2D+myotubes n=8 across N=3 3D+myotubes n=14 across n=3252D n=9 2D+myotubes n=8 3D+myotubes n=14One-way ANOVA with Tukey’s post-test
F5-S1C2D n=9 across N=3 2D+myotubes n=8 across N=3 3D+myotubes n=14 across n=3252D n=9 2D+myotubes n=8 3D+myotubes n=14One-way ANOVA with Tukey’s post-test
F5-S2C1DPE: n=35 across N=3 3DPE: n=45 across N=3 7DPE: n=45 across N=3Every 5 images is from 1 tissuen=125Simple linear regression
F5-S3A1DPE: n=916 across N=4 3DPE: n=980 across N=4 7DPE: n=737 across N=3251DPE: n=916 3DPE: n=980 7DPE: n=737–
F5-S3CPax7+/MyoD- 3DPE: n=12 across N=4 7DPE: n=11 across N=3 Pax7+/MyoD+ 3DPE: n=11 across N=4 7DPE: n=10 across N=325Pax7+/MyoD- 3DPE: n=12 7DPE: n=11 Pax7+/MyoD+ 3DPE: n=11 7DPE: n=10Unpaired t-tests
F5-S4BControl 1DPE: n=7 across N=3 3DPE: n=8 across N=3 7DPE: n=9 across N=3 Y-27632 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=325Control 1DPE: n=7 3DPE: n=8 7DPE: n=9 Y-27632 1DPE: n=9 3DPE: n=9 7DPE: n=9Unpaired t-tests for each individual timepoint
F5-S4CControl 1DPE: n=6 across N=2 3DPE: n=6 across N=2 7DPE: n=6 across N=2 Y-27632 1DPE: n=6 across N=2 3DPE: n=6 across N=2 7DPE: n=6 across N=225Control 1DPE: n=6 3DPE: n=6 7DPE: n=6 Y-27632 1DPE: n=6 3DPE: n=6 7DPE: n=6Unpaired t-tests for each individual timepoint
F5-S4DControl 1DPE: n=7 across N=3 3DPE: n=8 across N=3 7DPE: n=9 across N=3 Y-27632 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=325Control 1DPE: n=7 3DPE: n=8 7DPE: n=9 Y-27632 1DPE: n=9 3DPE: n=9 7DPE: n=9Unpaired t-tests for each individual timepoint
F5-S5AControl 1DPE: n=666 across N=3 3DPE: n=994 across N=3 7DPE: n=783 across N=3 Y-27632 1DPE: n=1,255 across N=3 3DPE: n=1,158 across N=3 7DPE: n=598 across N=325Control 1DPE: n=666 3DPE: n=994 7DPE: n=783 Y-27632 1DPE: n=1255 3DPE: n=1158 7DPE: n=598–
F5-S5BPax7+/MyoD- and Pax7+/MyoD+(Control 1DPE: n=7 across N=3 3DPE: n=8 across N=3 7DPE: n=9 across N=3 Y-27632 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3)25Pax7+/MyoD- and Pax7+/MyoD+ (Control 1DPE: n=7 3DPE: n=8 7DPE: n=9 Y-27632 1DPE: n=9 3DPE: n=9 7DPE: n=9)One-way ANOVA with Tukey’s post-test within each individual timepoints
F5-S5CPax7+/MyoD- and Pax7+/MyoD+(Control 1DPE: n=7 across N=3 3DPE: n=8 across N=3 7DPE: n=9 across N=3 Y-27632 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3)25Pax7+/MyoD- and Pax7+/MyoD+ (Control 1DPE: n=7 3DPE: n=8 7DPE: n=9 Y-27632 1DPE: n=9 3DPE: n=9 7DPE: n=9)One-way ANOVA with Tukey’s post-test within each individual timepoints
F5-S5EPax7+/MyoD- and Pax7+/MyoD+(Control 1DPE: n=7 across N=3 3DPE: n=8 across N=3 7DPE: n=9 across N=3 Y-27632 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3)25Control 1DPE: n=7 3DPE: n=8 7DPE: n=9 Y-27632 1DPE: n=9 3DPE: n=9 7DPE: n=9One-way ANOVA with Tukey’s post-test within each individual timepoints
F5-S5FPax7+/MyoD- and Pax7+/MyoD+(Control 1DPE: n=7 across N=3 3DPE: n=8 across N=3 7DPE: n=9 across N=3 Y-27632 1DPE: n=9 across N=3 3DPE: n=9 across N=3 7DPE: n=9 across N=3)25Pax7+/MyoD- and Pax7+/MyoD+ (Control 1DPE: n=7 3DPE: n=8 7DPE: n=9 Y-27632 1DPE: n=9 3DPE: n=9 7DPE: n=9)One-way ANOVA with Tukey’s post-test within each individual timepoints
F7-S1AYoung Day 1: n=6 across N=2 Day 3: n=6 across N=2 Young + wortmannin Day 1: n=6 across N=2 Day 3: n=6 across N=2 Aged Day 1: n=6 across N=2 Day 3: n=6 across N=2 Aged + wortmannin Day 1: n=6 across N=2 Day 3: n=6 across N=2104Young Day 1: n=6 Day 3: n=6 Young + wortmannin Day 1: n=6 Day 3: n=6 Aged Day 1: n=6 Day 3: n=6 Aged + wortmannin Day 1: n=6 Day 3: n=6One-way ANOVA with Tukey’s post-test comparing each experimental group at the 3 DPE timepoint
F7-S1BYoung n=2716 across N=2 Young + wortmannin n=565 across N=2 Aged n=4437 across N=2 Aged + wortmannin n=1897 across N=2104Young n=2716 Young + wortmannin n=565 Aged n=4437 Aged + wortmannin n=1897Outliers removed with the ROUT method (with Q=1%) and one-way ANOVA performed with Šidák’s post-test comparing pre-selected conditions
F7-S2BYoung n=9 across N=3 Young + wortmannin n=6 across N=2 Aged n=8 across N=3 Aged + ortmannin n=7 across N=325Young n=9 Young + wortmannin n=6 Aged n=8 Aged + wortmannin n=7One-way ANOVA with Tukey’s post-test

Additional files

MDAR checklist
https://cdn.elifesciences.org/articles/81738/elife-81738-mdarchecklist1-v2.docx

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