Journal: bioRxiv

Article Title: Rescue of aged muscle stem cell intrinsic quiescence defects by AKT inhibition revealed with a 3D biomimetic culture assay

doi: 10.1101/2022.06.15.496252

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) and nuclear fusion index (right-axis) 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 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 post-test, ** p=0.0033.

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: bioRxiv

Article Title: Rescue of aged muscle stem cell intrinsic quiescence defects by AKT inhibition revealed with a 3D biomimetic culture assay

doi: 10.1101/2022.06.15.496252

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 test for each individual timepoint comparing against the 500 MuSC condition, ** p=0.0025, 0.0051, 0.0029 **** p<0.0001.

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: bioRxiv

Article Title: Rescue of aged muscle stem cell intrinsic quiescence defects by AKT inhibition revealed with a 3D biomimetic culture assay

doi: 10.1101/2022.06.15.496252

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 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 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 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 (SAA: magenta) 2 days after a 4-hour 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 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

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: bioRxiv

Article Title: Rescue of aged muscle stem cell intrinsic quiescence defects by AKT inhibition revealed with a 3D biomimetic culture assay

doi: 10.1101/2022.06.15.496252

Figure Lengend Snippet: (A) Key for figure icons. (B-F) Line graphs of mononucleated DAPI + YFP + Pax7 + cell density at 1, 3 and 7 DPE (left) and pie charts showing the proportion of Ki67+/- cells at 7 DPE (right) for cells seeded into a 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=6-9 from N=2-3 independent biological replicates. Graphs display mean ± s.e.m.

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: bioRxiv

Article Title: Rescue of aged muscle stem cell intrinsic quiescence defects by AKT inhibition revealed with a 3D biomimetic culture assay

doi: 10.1101/2022.06.15.496252

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 DPE (top) and 7 DPI (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.

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

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: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

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 ) 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: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

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: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

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 ) 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: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

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 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: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

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: To quantify the metabolic activity of myotube templates, the MTS assay was used (abcam, #ab197010).

Techniques: Expressing