Organoid culture promotes dedifferentiation of mouse myoblasts into stem cells capable of complete muscle regeneration

Abstract

Experimental cell therapies for skeletal muscle conditions have shown little success, primarily because they use committed myogenic progenitors rather than true muscle stem cells, known as satellite cells. Here we present a method to generate in vitro-derived satellite cells (idSCs) from skeletal muscle tissue. When transplanted in small numbers into mouse muscle, mouse idSCs fuse into myofibers, repopulate the satellite cell niche, self-renew, support multiple rounds of muscle regeneration and improve force production on par with freshly isolated satellite cells in damaged skeletal muscle. We compared the epigenomic and transcriptional signatures between idSCs, myoblasts and satellite cells and used these signatures to identify core signaling pathways and genes that confer idSC functionality. Finally, from human muscle biopsies, we successfully generated satellite cell-like cells in vitro. After further development, idSCs may provide a scalable source of cells for the treatment of genetic muscle disorders, trauma-induced muscle damage and age-related muscle weakness.

Fig. 1. Mouse myoblasts can form SkMOs that share in vitro characteristics with satellite cells.

a, Schematic of the isolation and expansion of mouse myoblasts in vitro followed by the formation, growth and maturation of an SkMO. b, Immunofluorescent images of a late-stage SkMO stained with Pax7 (green), MyHC (red) and nuclear counterstain Hoechst (blue). Scale bars, 100 µm. c, Immunostaining of myoblasts and late-stage SkMOs for MyoD (red), Pax7 (green) and Hoechst (blue). Scale bars, 20 µm. d, Percentage of Pax7⁺MyoD⁺ cells in myoblasts and late-stage SkMOs. Box bounds represent s.e.m.; red line represents mean (n = 3, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction. P value: Myo versus SkMO 3.37 × 10⁻⁵; ***P < 0.001. e, Immunofluorescent images from EdU pulse chase experiments over 48 h comparing myoblasts, quiescent satellite cells and late-stage SkMO cells. Images show Pax7 (green), EdU (red) and Hoechst (blue). Scale bars, 10 µm. f, Percentage of Pax7⁺EdU⁺ cells in each cell condition. Box bounds represent s.e.m.; red line represents mean (satellite cell n = 4, myoblast n = 3, SkMO n = 3, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction between samples. P values: SC versus SkMO 0.205, SC versus Myo 2.0 × 10⁻⁶; Myo versus idSC 3.5 × 10⁻⁵; ***P < 0.001. g, Representative images of myoblasts, SkMO-derived GFP⁺ cells and quiescent satellite cells immediately after FACS isolation. Scale bars, 10 µm. h, Comparisons of cell diameter in proliferative myoblasts, SkMO-derived GFP⁺ cells and quiescent satellite cells. Box bounds represent s.e.m.; red line represents mean (n = 3, biological replicates with ≥100 cells per replicate). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction between samples. P values: Myo versus SC 2.22 × 10⁻⁴; Myo versus SkMO 8.98 × 10⁻⁴; SkMO versus SC 1.7 × 10⁻⁴; ***P < 0.001. NS, not significant; Myo, myoblast; SC, satellite cell.

Fig. 2. idSCs share key transcriptional similarities with satellite cells.

a, Heatmap depicting genes key to satellite cells, early activation and stress response, quiescence, cell surface receptors, ECM, secreted growth factors and Notch pathway genes colored based on z-score from high (red) to low (blue) (n > 4). b, Venn diagram depicting significant genes (FDR ≤ 0.1) among myoblasts, idSCs and satellite cells. c, Volcano plots depicting log₂ fold change in gene expression between satellite cells versus myoblasts (bottom left), idSCs versus myoblasts (top right), and idSCs versus satellite cells (bottom right). Genes that are significant (FDR ≤ 0.1) are shown in red. d, KEGG pathways identified based on comparing differentially expressed genes among myoblasts, idSCs and satellite cells and visualized based on significance using a hypergeometric test (−log(P value)). Highlighted pathways are directly referenced in the text. Myo, myoblast; SC, satellite cell.

Fig. 3. Key regulatory regions in myoblasts that define the myogenic program are silenced during idSC generation.

a, Heatmap depicting dynamic patterns of change in chromatin accessibility that occur between proliferative myoblasts and idSCs at early-stage (day 10), mid-stage (day 20) or late-stage (day 30) culture timepoints and freshly isolated satellite cells. Clustering of peaks into three broad states: myoblast specific, transition state specific and satellite cell specific. Boxed region highlights myoblast specific peaks in an open configuration remodeled into closed chromatin during idSC formation. b, Clade analysis with associated GO BP terms generated by GREAT based on an unbiased dendogram depicting similarities in chromatin accessibility across sample types. c, Heatmap depicting dynamic patterns of change in chromatin accessibility conserved between late-stage idSCs and satellite cells relative to myoblasts. d, Associated GO Cellular Component terms for genes with cis-regulatory peaks conserved between late-stage idSCs and satellite cells using a binomial test. e, Associated GO BP terms for genes with cis-regulatory bound E-box sites specific to myoblasts using a binomial test. f, Footprinting of E-box sites bound in myoblasts across idSC samples and satellite cells. Example shows progressive loss in accessibility over time in cultured idSCs, similar to satellite cell footprinting. SC, satellite cell.

Fig. 4. idSCs engraft, repopulate and self-renew in vivo.

a, Experimental schematic outlining the transplant and analysis of myoblasts or idSCs into regenerating TA muscle of NSG mice. b, Quantification of the average radiance (photons cm⁻² s⁻¹) across a standardized region of interest (ROI) superimposed over NSG hindlimbs over a 21-d timecourse after transplant of 10,000 myoblasts (yellow) or idSCs (green). Data represent mean ± s.e.m (n = 97 day 7; n = 102 day 14; n = 92 day 21, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction. P values: day 7 0.011; day 14 5.23 × 10⁻⁶; day 21 5.40 × 10⁻⁵; *P < 0.05, ***P < 0.001. c, Representative immunofluorescence micrograph depicting engraftment after the transplant of nGFP⁺tdTomato⁺ myoblasts or idSCs into newly regenerated muscle of NSG mice. Images were stained with laminin (green), tdTomato (red) and Hoechst (blue). Scale bar, 100 µm. d, Quantification of the percentage of tdTomato⁺ fibers per muscle cross-section after transplant of either myoblasts or idSCs. Box bounds represent s.e.m.; bar represents mean (n = 5, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction. P value: idSC versus Myo 6.70 × 10⁻⁵; ***P < 0.001. e, Representative immunofluorescent micrograph depicting the ability of donor-derived cells to repopulate the satellite cell niche and express Pax7 (green), tdTomato (red) and Hoechst (blue). Arrowheads represent sublaminar Pax7⁺tdTomato⁻ cells within NSG mice. Arrows indicate sublaminar Pax7⁺tdTomato⁺ cells. Scale bar, 100 µm. f, Quantification of the percent of sublaminar Pax7⁺tdTomato⁺ cells relative to the total Pax7⁺ cells per section. Percent double-positive cells was derived from an average total of Pax7⁺tdTomato⁺ to Pax7⁺ cells quantified per replicate (shown in panel). Box bounds represent s.e.m.; bar represents mean (n = 5, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction. P value: idSC versus Myo 7.24 × 10⁻³; **P < 0.01. g, 63-d longitudinal BLI study examining engraftment of 10,000 idSCs or myoblasts, followed by re-injury of muscle at 21 d and 42 d after transplant. Data represent mean ± s.e.m. (n = 18, days 21, 28, 35 and 42; n = 15, days 44, 49, 56 and 63; n = 3, day 23, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction. P values: day 21, 0.005; day 23, 0.077; day 28, 0.001; day 35, 0.001; day 42, 0.015; day 44, 0.019; day 49, 0.024; day 56, 0.044; day 63, 0.030; *P < 0.05, **P < 0.01, ***P < 0.001. h, Immunofluorescence micrograph of a TA cross-section before the second round of re-injury (day 42). tdTomato⁺ (red) and laminin (green). Scale bars, 500 µm. i, Percentage of tdTomato⁺ fibers per muscle section (n = 3, biological replicates). Box bounds represent s.e.m.; red line represents mean. Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction. P value: Myo versus idSC 0.012; *P < 0.05. Myo, myoblast.

Fig. 5. idSCs engraft, repopulate and self-renew in vivo in a dystrophin-deficient mouse model.

a, Schematic summarizing isolation and transplant timelines. b, Representative immunofluorescence micrograph depicting engraftment after the transplant of 10,000 FACS-sorted nGFP⁺ myoblasts, idSCs or freshly isolated satellite cells into irradiated (IRR) (13 Gy) TA muscles of mdx^5cv mice. Images were stained with dystrophin (green), laminin (red) and Hoechst (blue). Scale bar, 100 µm. c, Quantification of the percentage of dystrophin⁺ fibers relative to the total laminin⁺ fibers per muscle cross-section. Box bounds represent s.e.m.; bar represents mean (day 21: n = 8 Myo, n = 10 idSC, n = 7 SC; day 42: n = 9 idSC, n = 4 SC, biological replicates) One-way ANOVA with Tukey HSD analysis conducted at day 21 and day 42 between samples. Day 21 ANOVA P value 6.82 × 10⁻⁶, Tukey HSD adjusted P values: day 21 idSC versus Myo 5.48 × 10⁻⁶, day 21 idSC versus SC 0.292, day 21 Myo versus SC 6.26 × 10⁻⁴; day 42 ANOVA P value 0.049. *P < 0.05, **P < 0.01, ***P < 0.001. d, Representative immunofluorescent micrograph depicting the ability of donor-derived cells to repopulate the satellite cell niche and express Pax7 (green), nGFP (red), laminin (white) and Hoechst (blue). Arrowheads represent sublaminar Pax7⁺nGFP⁻ (endogenous) satellite cells within mdx^5cv mice. Arrows indicate sublaminar Pax7⁺nGFP⁺ cells (donor derived). Scale bars, 100 µm. e, Quantification of the percent of sublaminar Pax7⁺nGFP⁺ cells per section. Box bounds repreent s.e.m.; bar represents mean (day 21 n = 8 Myo, n = 6 idSC, n = 7 SC; day 42 n = 8 idSC, n = 3 SC, biological replicates). One-way ANOVA with Tukey HSD analysis conducted at day 21 and day 42 between samples. Day 21 ANOVA P value 7.31 × 10⁻⁴, Tukey HSD adjusted P values: idSC versus Myo 4.73 × 10⁻³, idSC versus SC 0.91, Myo versus SC 1.26 × 10⁻³. Day 42 ANOVA P value 0.035. *P < 0.05, **P < 0.01, ***P < 0.001. Raw counts for the number of Pax7⁺nGFP⁺/total Pax7⁺ are displayed at the top of the panel. f, Representative immunofluorescent micrograph depicting Pax7, nGFP, Ki67 and Hoechst. Arrows represent Pax7⁺nGFP⁺ cells (donor derived). Scale bars, 25 µm. g, Quantification of the percent of sublaminar Pax7⁺nGFP⁺Ki67⁻ cells per section. Data represent mean ± s.e.m. (n = 8 idSC; n = 5 SC, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction between samples. P value: SC versus idSC 0.166. Raw counts for the number of Pax7⁺nGFP⁺Ki67⁻/total Pax7⁺nGFP⁺ are displayed at the top of the panel. HSD, honestly significant difference; NS, not significant; SC, satellite cell.

Fig. 6. idSCs are similar to satellite cells in their capacity to generate functional muscle after transplantation into regenerating muscle.

a, Experimental schematic outlining the transplant of 10,000 nGFP⁺ cells (myoblasts, idSCs or freshly isolated satellite cells) into irradiated (IRR) and subsequently damaged TA muscles of NSG mice. b, Specific maximum tetanic force assessed on TA muscles 21 d after CTX damage. Undamaged muscle and sham transplants gave rise to specific force values of 278 ± 20.24 kN × m⁻² and 8 ± 2.47 kN × m⁻², respectively. Box bounds represent s.e.m.; red line represents mean (n = 6 undamaged, n = 8 sham, n = 15 Myo, n = 12 SC, n = 12 idSC, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction between samples. P values: undamaged versus idSC 2.56 × 10⁻⁷; undamaged versus SC 9.02 × 10⁻⁵; Myo versus idSC 1.37 × 10⁻⁹; Myo versus SC 6.88 × 10⁻¹³; SC versus idSC 0.0405; Myo versus sham 0.818; *P < 0.05, ***P < 0.001. c, Stitched hematoxylin and eosin micrographs of cross-sectioned TA muscle 21 d after cell transplant. Scale bars, 100 µm. d, Total muscle fibers per section across samples. Box bounds represent s.e.m.; red line represents mean (n = 3, biological replicates). Student’s two-tailed t-test, unpaired with equal variance assumed and no multiple testing correction between samples. P values: undamaged versus idSC 0.358; undamaged versus SC 0.0175; Myo versus idSC 9.22 × 10⁻⁶; SC versus idSC 0.0897; Myo versus SC 8.39 × 10⁻⁵; Myo versus sham 0.804; *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant; SC, satellite cell.