Muscle Stem Cells Exhibit Distinct Clonal Dynamics in Response to Tissue Repair and Homeostatic Aging

Abstract

The clonal complexity of adult stem cell pools is progressively lost during homeostatic turnover in several tissues, suggesting a decrease in the number of stem cells with distinct clonal origins. The functional impact of reduced complexity on stem cell pools, and how different tissue microenvironments may contribute to such a reduction, are poorly understood. Here, we performed clonal multicolor lineage tracing of skeletal muscle stem cells (MuSCs) to address these questions. We found that MuSC clonal complexity is maintained during aging despite heterogenous reductions in proliferative capacity, allowing aged muscle to mount a clonally diverse, albeit diminished, response to injury. In contrast, repeated bouts of tissue repair cause a progressive reduction in MuSC clonal complexity indicative of neutral drift. Consistently, biostatistical modeling suggests that MuSCs undergo symmetric expansions with stochastic fate acquisition during tissue repair. These findings establish distinct principles that underlie stem cell dynamics during homeostatic aging and muscle regeneration.

Keywords: aging; biostatistical modeling; clonal behavior; clonal complexity; functional heterogeneity; homeostasis; multicolor lineage tracing; regeneration; skeletal muscle; stem cells.

Figure 1. MuSC contribution to homeostatic aging is robust and polyclonal

(A-B) Scheme and identification of FP⁺ myofibers in composite images following MuSC labeling and long-term lineage tracing in Pax7-CreER™; R26R^Brainbow2.1 muscles with age. Scale bar, 50 µm. (C-D) Quantification of myofiber CSA and the percentage of myofibers expressing 0, 1, or 2 or more FPs in young and aged muscles (n = 5). Data are represented as average ± SEM (***P < 0.001, *P < 0.05). See also Figure S1.

Figure 2. Functional MuSC heterogeneity, but not clonal complexity, is reduced with age

(A) Scheme FP⁺ MuSC-derived clonal labeling in young and aged muscles upon single myofibers cultured in suspension and 3 d post-BaCl₂ injury. (B-C) Images and quantification of FP⁺ myofiber-associated MuSC clone size and the percentage of Pax7⁺ cells per clone isolated from young and aged muscles after 3 d in suspension culture. Scale bar, 50 µm (n = 53-64 clones). (D-E) Composite images and quantification of FP⁺ MuSC-derived clone size and the percentage of Myod⁺ cells per clone in young and aged muscles 3 d post-BaCl₂ injury. Scale bar, 50 µm (n = 62-81 clones). (F-G) Scheme and identification of FP⁺ myofibers in composite images of young and aged muscles 25 d post-BaCl₂ injury. Scale bar, 50 µm. (H-I) Quantification of myofiber CSA and the percentage of myofibers expressing any number of FPs in young and aged muscles 25 d post-BaCl₂ injury (n = 5). Data are represented as average ± SEM (***P < 0.001, **P < 0.01, *P < 0.05; student's t-test unless otherwise indicated). See also Figure S2.

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 d post-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-D) Scheme and identification of FP⁺ myofibers in composite images of young muscles following serial BaCl₂ injury. Scale bar, 50 µm. (E-F) Quantification of myofiber CSA and the percentage of myofibers expressing any number of FPs in young muscles following serial BaCl₂ injury (n = 5). Data are represented as average ± SEM (**P < 0.01, *P < 0.05). See also Figure S3.

Figure 4. MuSCs follow a model of population asymmetry and neutral drift with repeated injury

(A) Composite images of GFP⁺ myonuclei in regenerated muscles following serial BaCl₂ injury. Scale bar, 100 µm. (B-C) Quantification of GFP⁺ myofiber cluster density and the number of regenerated myofibers per cluster following serial BaCl₂ injury (n = 5). (D) Cumulative, scaled cluster size distribution following serial BaCl₂ injury (199-446 clusters). (E-F) Quantification of nearest neighbor index and cumulative distribution frequency derived from spatial analyses of GFP⁺ nuclei following serial BaCl₂ injury (n = 5). Data are represented as average ± SEM (***P < 0.001, **P < 0.01, *P < 0.05). See also Figure S4.