Volume 22, Issue 1, Pages 119-127.e3 (January 2018) Muscle Stem Cells Exhibit Distinct Clonal Dynamics in Response to Tissue Repair and Homeostatic Aging  Matthew T. Tierney, Michael J. Stec, Steffen Rulands, Benjamin D. Simons, Alessandra Sacco  Cell Stem Cell  Volume 22, Issue 1, Pages 119-127.e3 (January 2018) DOI: 10.1016/j.stem.2017.11.009 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Stem Cell 2018 22, 119-127.e3DOI: (10.1016/j.stem.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 MuSC Contribution to Homeostatic Aging Is Robust and Polyclonal (A and B) Scheme (A) and identification of FP+ myofibers (B) in composite images following MuSC labeling and long-term lineage tracing in Pax7-CreERTM;R26RBrainbow2.1 muscles with age. Scale bar, 50 μm. (C and D) Quantification of myofiber CSA (C) and the percentage of myofibers expressing 0, 1, or 2 or more FPs (D) in young and aged muscles (n = 5). Data are presented as mean ± SEM (∗∗∗p < 0.001, ∗p < 0.05). See also Figure S1. Cell Stem Cell 2018 22, 119-127.e3DOI: (10.1016/j.stem.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Functional MuSC Heterogeneity, but Not Clonal Complexity, Is Reduced with Age (A) Scheme of FP+ MuSC-derived clonal labeling in young and aged muscles upon single myofibers cultured in suspension and 3 days after BaCl2 injury. (B and C) Images (B) and quantification of FP+ myofiber-associated MuSC clone size and the percentage of PAX7+ cells per clone (C) isolated from young and aged muscles after 3 days in suspension culture. Scale bar, 50 μm (n = 53–64 clones). (D and E) Composite images (D) and quantification of FP+ MuSC-derived clone size and the percentage of MYOD+ cells per clone (E) in young and aged muscles 3 days after BaCl2 injury. Scale bar, 50 μm (n = 62–81 clones). (F and G) Scheme (F) and identification of FP+ myofibers (G) in composite images of young and aged muscles 25 days after BaCl2 injury. Scale bar, 50 μm. (H and I) Quantification of myofiber CSA (H) and the percentage of myofibers expressing any number of FPs (I) in young and aged muscles 25 days after BaCl2 injury (n = 5). Data are presented as mean ± SEM (∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05; Student’s t test unless otherwise indicated). See also Figure S2. Cell Stem Cell 2018 22, 119-127.e3DOI: (10.1016/j.stem.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Symmetric Expansion and Stochastic Fate Acquisition Precedes a Decline in MuSC Clonal Complexity under Constant Regenerative Pressure (A) Young and aged MuSC cumulative clone size distributions and model predictions 3 days after BaCl2 injury. Shaded area denotes 95% Kolmogorov-Smirnov confidence intervals of empirical distribution. (B) Scheme depicting young and aged MuSC behavior during the early regenerative phase. (C and D) Scheme (C) and identification of FP+ myofibers (D) in composite images of young muscles after serial BaCl2 injury. Scale bar, 50 μm. (E and F) Quantification of myofiber CSA (E) and the percentage of myofibers expressing any number of FPs (F) in young muscles after serial BaCl2 injury (n = 5). Data are presented as mean ± SEM (∗∗p < 0.01, ∗p < 0.05). See also Figure S3. Cell Stem Cell 2018 22, 119-127.e3DOI: (10.1016/j.stem.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 MuSCs Follow a Model of Population Asymmetry and Neutral Drift with Repeated Injury (A) Composite images of GFP+ myonuclei in regenerated muscles after serial BaCl2 injury. Scale bar, 100 μm. (B and C) Quantification of GFP+ myofiber cluster density (B) and the number of regenerated myofibers per cluster (C) after serial BaCl2 injury (n = 5). (D) Cumulative, scaled cluster size distribution after serial BaCl2 injury (199–446 clusters). (E and F) Quantification of nearest-neighbor index (E) and cumulative distribution frequency (F) derived from spatial analyses of GFP+ nuclei after serial BaCl2 injury (n = 5). Data are presented as mean ± SEM (∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05). See also Figure S4. Cell Stem Cell 2018 22, 119-127.e3DOI: (10.1016/j.stem.2017.11.009) Copyright © 2017 Elsevier Inc. Terms and Conditions