Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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 myotube templates labelled for sarcomeric α-actinin (SAA; magenta) at days 2, 5, 10, 14, 16, and 18 of culture. Scale bar, 1 mm. ( C ) Representative confocal image of myotubes at day 5 labelled with DAPI (cyan) and SAA (magenta). Scale bar, 50 µm. ( D ) Quantification of SAA area coverage (left axis; black line) and nuclear fusion index (right axis; grey line) of myotube templates at days 2, 5, 10, 14, 16, and 18 of culture. n=9–16 across N=3–6 independent biological replicates. Graph displays mean ± s.e.m.; one-way ANOVA with Tukey’s post-test, minimum ***p=0.002 (SAA coverage), ****p˂0.0001 (nuclear fusion index). ( E ) Optical density (OD) at 490 nm of media after myotube template incubation with MTS assay reagent on days 2, 5, 10, 14, 16, and 18 of culture. n=9–12 across N=3–4 independent biological replicates. Graph displays mean ± s.e.m.; one-way ANOVA with Tukey’s post-test, **p=0.0033. Raw data available in . Figure 1—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques: Incubation, MTS Assay

Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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 ) Quantification of SAA area coverage of myotube templates at day 5 of differentiation using 10,000, 25,000, or 50,000 cells. n=12 across N=3 independent biological replicates. Graph displays technical replicates with mean ± s.e.m.; one-way ANOVA with Tukey’s post-test, ***p=0.0003, ****p˂0.0001. Raw data available in . Figure 1—figure supplement 1—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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) vs. 2D monolayer (bottom row). In ( A ), white arrow shows myotube detachment site in 2D cultures and scale bar, 1 mm. In ( B ), white arrows show detached myotubes, termed ‘myoballs’ and scale bar, 50 µm. ( C ) Bar graph displaying SAA area coverage at days 5, 8, and 12 of differentiation in 3D vs. 2D. n=6–9 across N=3 independent biological replicates, graph displays mean ± s.e.m. with the individual technical replicates; one-way ANOVA with Tukey’s post-test, *p=0.0264, 0.0429. ( D ) Quantification of myoballs per 100 µm 2 at days 5, 8, and 12 of differentiation in 3D vs. 2D. n=6–9 across N=3 independent biological replicates, graph displays mean ± s.e.m. with the individual technical replicates; one-way ANOVA with Tukey’s post-test, *p=0.0343, **p=0.0081, 0.0081, 0.0053, 0.0029. ( E ) Bar graph showing fraction of α-actinin skeleton classified as Z-lines by actin guided segmentation (Z-line fraction) at days 5, 8 and 12 of differentiation in 3D vs. 2D. n=6–9 across N=3 independent biological replicates, graph displays mean ± s.e.m. with the individual technical replicates; one-way ANOVA with Tukey’s post-test, *p=0.0226, **p=0.0020, 0.0081. ( F ) Bar graph with mean continuous Z-line length at days 5, 8, and 12 of differentiation in 3D vs. 2D. n=6–9 across N=3 independent biological replicates, graph displays mean ± s.e.m. with the individual technical replicates; one-way ANOVA with Tukey’s post-test, ns. ( G ) Bar graph of mean sarcomere length at days 5, 8, and 12 of differentiation in 3D vs. 2D. n=6–9 across N=3 independent biological replicates, graph displays mean ± s.e.m. with the individual technical replicates; one-way ANOVA with Tukey’s post-test, *p=0.0356, **p=0.0097, 0.0025, ***p=0.0003. Raw data available in . Figure 1—figure supplement 2—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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: white, white arrows) at 1 day post-engraftment (DPE) onto myotube templates. Scale bar, 50 µm. ( C ) Quantification of the percentage Pax7 + cells in the DAPI + YFP + population at 1 DPE after engraftment with CAG-EYFP reporter MuSCs. n=11 across N=3 independent biological replicates. Graph displays mean ± s.e.m. of technical replicates. Raw data available in . Figure 2—figure supplement 1—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques: Flow Cytometry, Muscles

Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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 (phalloidin: magenta) with engrafted MuSCs (YFP: yellow, Pax7: white, white arrows) at 1, 3, and 7 days post-engraftment (DPE). Scale bar, 50 µm. ( C ) Representative confocal image of a donor MuSC (DAPI: cyan, YFP: yellow, Pax7: white) indicated with a white arrow, and myotubes (phalloidin: magenta) at 7 DPE. Scale bar, 20 µm. ( D ) Quantification of mononuclear DAPI + YFP + Pax7 + cell density per mm 2 at 1, 3, and 7 DPE across different starting MuSC engraftment numbers (200, 500, 1500, and 2500). n=9–15 across N=3–5 independent biological replicates. Graph displays mean ± s.e.m.; one-way ANOVA with Dunnet’s test for each individual timepoint comparing against the 500 MuSC condition, **p=0.0025, 0.0051, 0.0029, ****p˂0.0001. Raw data available in . Figure 2—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques: Isolation

Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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 DPE (bottom). Scale bar, 20 µm. ( B ) Stacked bar graph showing proportions of c-FOS ± cells at 1, 3, and 7 DPE in the DAPI + YFP + Cav-1 + population. n=9 across N=3 independent biological replicates. Graph displays mean ± s.e.m. for c-FOS + and c-FOS - ; one-way ANOVA with Tukey’s post-test comparing the FOS - proportions of each timepoint, ****p˂0.0001. ( C ) Stacked bar graph showing proportions of Ki67 ± cells at 1, 3, and 7 DPE in the DAPI + YFP + Pax7 + population. n=10–11 across N=3–4 independent biological replicates. Graph displays mean ± s.e.m. for Ki67 + and Ki67 - ; one-way ANOVA with Tukey’s post-test comparing the Ki67 - proportions of each timepoint, ****p˂0.000.1. ( D ) Timeline of EdU/Ki67 co-labelling experiment (made with BioRender). ( E ) Stacked bar graph showing proportions of EdU ± cells at 7 DPE in the DAPI + YFP + Ki67 - mononuclear cell population. n=15 across N=5 independent biological replicates. Graph displays mean ± s.e.m. for EdU + and EdU - . ( F ) Representative confocal stitched images of myotube templates (sarcomeric α-actinin (SAA): magenta) 2 days after a 4 hr exposure to the physiological salt solution (PSS) control or a 2.4% barium chloride (BaCl 2 ) solution. Scale bar, 1 mm. ( G ) Proportion of Ki67 ± cells at 2 days post-injury (DPI) in the DAP + YFP + Pax7 + population. n=16, 18 across N=5, 6 biological replicates. Graph displays mean ± s.e.m. for Ki67 + and Ki67 - ; unpaired t-test of the Ki67 - proportions of both conditions, ****p˂0.0001. Raw data available in . Figure 3—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques: Control

Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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 DPE (right) for cells seeded into a two-dimensional (2D) microwell with a Geltrex coating ( B ), engrafted into 3D myotube templates on day 5 ( C ) vs. day 0 ( D ) of differentiation. Additional comparisons include engraftment into a 3D cellulose-reinforced extracellular matrix (ECM) hydrogel on day 5 ( E ), or onto a 2D monolayer of myotubes with a Geltrex undercoating on day 5 of differentiation ( F ). n=6–15 from N=2–3 independent biological replicates. Graphs display mean ± s.e.m. ( G ) Representative confocal images of YFP + (yellow) donor cells (DAPI: cyan) at 7 DPE engrafted in 2D, 3D with myotubes (day 5), 3D with myocytes (day 0), 3D with cellulose-reinforced ECM or with a 2D monolayer of myotubes. Cells are also labelled for Ki67 (magenta) and Pax7 (white) where Ki67 - Pax7 + cells are indicated with white arrows, Ki67 + Pax7 + with grey arrows, and Ki67 + Pax7 - with magenta arrows. Scale bar, 50 µm. Raw data available in . Figure 4—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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 differentiation), and immunolabelled for YFP (yellow). Scale bar, 50 µm. ( B ) Quantification of percentage of tissue area covered by YFP signal at 7 DPE when 500 MuSCs are engrafted on day 0 vs. day 5. n=11, 15 from N=4, 5 independent biological replicates. Graph displays mean ± s.e.m. with individual technical replicates; unpaired t-test, **p=0.0012. Raw data available in . Figure 4—figure supplement 1—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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 on a Geltrex undercoating (middle), or engrafted into 3D myotube templates at day 5 of differentiation (right). Scale bar, 20 µm. ( B ) Bar plot showing the average max/min Feret diameter of the mononuclear DAPI YFP Pax7 + population at 7 days post-engraftment (DPE) engrafted in 2D, 2D myotube co-culture, or in 3D myotube template co-culture. n=8–15 across N=3 independent biological replicates. Graph displays mean ± s.e.m. with individual technical replicates; one-way ANOVA with Tukey’s post-test, ****p˂0.0001. ( C ) Bar plot showing the average max/min Feret diameter of the mononuclear DAPI + YFP + Pax7 + population at 7 DPE engrafted in in 2D, 2D myotube co-culture, or in 3D myotube template co-culture. n=8–15 across N=3 independent biological replicates. Graph displays mean ± s.e.m. with individual technical replicates; one-way ANOVA with Tukey’s post-test, ***p=0.0005, ****p˂0.0001. Raw data available in . Figure 5—figure supplement 1—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques: Co-Culture Assay

Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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 apical side. Cells are associated with myotubes that were visualized using the background signal (white dotted lines). Scale bars, 20 µm.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques:

Journal: eLife

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

doi: 10.7554/eLife.81738

Figure Lengend Snippet: ( 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 projection (white arrowhead). Scale bar, 20 µm. ( B ) Representative confocal images of a mononuclear donor cell (DAPI: cyan, YFP: yellow) at 1 day post-engraftment (DPE) (top) and 7 DPE (middle and bottom) expressing integrin α-7 (magenta) and M-cadherin (white). Middle inset image channels are separated to produce the bottom images to highlight the polarization of integrin α-7 and M-cadherin (white arrow) to basal and apical orientations, respectively (dotted lines). Scale bars, 20 µm. ( C ) Bar plot showing the percentage of mononuclear DAPI + YFP + cells with N-cadherin + cytoplasmic projections at 7 DPE. n=8 across N=3 independent biological replicates. Graph displays mean ± s.e.m. with individual technical replicates. ( D ) Bar plot showing the percentage of mononuclear DAPI + YFP + cells with polarized integrin α-7 (Iα7)/M-cadherin expression at 7 DPE. n=8 across N=3 independent biological replicates. Graph displays mean ± s.e.m. with individual technical replicates. Raw data available in . Figure 6—source data 1. Raw data for . Data for subpanels separated into tabs.

Article Snippet: One day prior to seeding myotube templates, black 96-well clear bottom plates (PerkinElmer, #6055300) were coated with 5% pluronic acid (Sigma-Aldrich, #P2443) and incubated overnight at 4°C.

Techniques: Expressing