A dual-color PAX7 and MYF5 in vivo reporter to investigate muscle stem cell heterogeneity in regeneration and aging

Abstract

Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5^Low MuSCs and skews the stem cell pool toward MYF5^High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.

Keywords: MYF5; PAX7; Skeletal muscle; aging; muscle stem cells; quiescence; regeneration; satellite cells; self-renewal; stemness.

Figure 1

Generation and characterization of StemRep mice (A and B) Genetic constructs of (A) Pax7-ZsGreen1-NLS and (B) Myf5-E2Crimson-NLS. Representation of the wild-type allele (top), targeted allele (middle), and knockin allele after flippase-FRT recombination (bottom). (C) Representative flow cytometry plots of MuSCs (red) derived from StemRep mice and the associated CD11b-/CD31-/CD45-/LY-6A/E−/CD34+/ITGA7+ antigen signature. (D and E) Post-sort representative images of cytospin preparations (D) and quantification (E) of MuSCs isolated based on nuclear ZsGreen1 fluorescence that stained positive using antibodies for PAX7. ZsGreen1 reporter, green; PAX7, red; Nuclei (Hoechst), blue. Scale bar, 50 μm. n = 4 mice. Data presented are means ± SEM. ns = not significant in a two-tailed unpaired Student’s t test (E).

Figure 2

Ex vivo and in vivo comparative characterization of MYF5^High and MYF5^Low MuSCs (A) Flow cytometry-based isolation strategy for MYF5^High and MYF5^Low MuSCs. (B) Representative images of MYF5^High and MYF5^Low cells in cytospin preparations immediately after sorting. Scale bar, 100 μm. (C) Histogram showing the proportions of cells positive for either PAX7-ZsGreen1 or MYF5-E2Crimson, or both in isolated MYF5^High and MYF5^Low fractions. (D) Distribution of MYF5^High and MYF5^Low cells according to E2Crimson intensity following isolation. A total of 1,000 cells per subpopulation were analyzed. (E and F) Average intensity of E2Crimson (E) and ZsGreen1 (F) fluorescence in each subpopulation. (G–I) Quantification of cells positive for PAX7-ZsGreen1 endogenous fluorescence and immunolabeled MYF5-E2Crimson on healthy muscle tissue sections. (G) Representative images and (H) quantification of the number of PAX7-ZsGreen1 and (I) of the mean intensity of immunolabeled E2Crimson (MYF5) signal on tibialis anterior cross-sections in uninjured muscle from StemRep mice. Scale bar, 50 μm. The number of MuSCs in vivo was estimated by averaging all PAX7-ZsGreen1-positive cells from 2 complete muscle sections per mouse. n = 4 mice. Data presented are means ± SEM; ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 in a two-tailed unpaired Student’s t test (C, E, F, H, I).

Figure 3

Distinct clonal and functional properties of MYF5^High and MYF5^Low MuSCs (A–C) Representative images (A), nuclei count (B), and size distribution of clones from freshly isolated MYF5^High and MYF5^Low cells after 10 days of culture (C). n = 6 × 96-well plates from a pool of 4 mice. Scale bar, 1 mm. (D) Representative pictures of PAX7, MYOD, and Myogenin expression in MYF5^High and MYF5^Low cells after 48 h of culture. Scale bars, 50 μm. (E–G) Quantification of PAX7 (E), MYOD (F), and Myogenin (G) over a 3-day time course. n = 4 mice. (H–J) Representative pictures (H) and quantification of fusion capacity (I) and myotube size (J) of MYF5 subpopulations. Myotube area was defined as the area positive for the myosin heavy chain (MyHC). Scale bar, 100 μm. n = 4 mice. Data presented are means ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 in a two-tailed unpaired Student’s t test for each condition and/or time point (B, C, D, E, F, G, I, J).

Figure 4

MYF5^Low MuSCs activate slower than MYF5^High cells (A) Principal component analysis of MYF5^High and MYF5^Low MuSCs isolated from male and female StemRep mice. Each datapoint represents 50,000 MuSCs isolated from a pool of 4 animals. (B) Heatmap of differentially expressed genes between the MYF5^High and MYF5^Low transcriptomes. (C and D) Differential expression of selected muscle- (C) and cell cycle-related genes (D) between MYF5 fractions in the male datasets. (E–G) Representative images (E) and quantification of nuclei (F) and EdU-positive cells (G) in MYF5^High and MYF5^Low subpopulations over a 3-day time course. (H–J) Cell cycle analysis of MYF5^Low (H) and MYF5^High (I) populations by flow cytometry and corresponding quantification of cell proportion within each cell cycle phase (J). Scale bar, 50 μm. n = 4 mice. Data represent means ± SEM; ^∗p < 0.05, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 in a two-tailed unpaired Student’s t test for each condition and/or time point (C, D, F, G, J).

Figure 5

MYF5^Low cells are deeply quiescent and exhibit increased self-renewal compared to MYF5^High cells ex vivo (A–D) qPCR of canonical quiescence genes in MYF5 subpopulations. n = 8 mice. (E–G) Representative images at 36 h (E) and ex vivo quantification at 18 (F) and 36 h (G) post-sort of asymmetric segregation of MYOD in MYF5 cells. Scale bar, 50 mm. n = 4 mice. Data represent means ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001 in a two-tailed unpaired Student’s t test for each population (A, B, C, D, F, G).

Figure 6

Characterization of MYF5^High and MYF5^Low MuSC subpopulations during muscle regeneration (A and B) Flow cytometry analysis of PAX7-ZsGreen1 (A) and MYF5-E2Crimson (B) fluorescence intensity dynamics in MuSCs isolated from quadriceps under uninjured conditions and at 3, 7, 14, and 21 dpi. (C and D) Quantification (C) and representative images (D) of MuSC subpopulations from tibialis anterior histological cross-sections at multiple stages of regeneration. Scale bar, 50 μm. n = 4 mice per group, each datapoint represents one mouse. Data presented are means ± SEM.

Figure 7

Aging primarily affects MYF5^Low MuSCs (A) Representative flow cytometry profiles of MuSCs isolated from adult (4 m.o., left) and aged (22 m.o., right) StemRep mice. (B) Flow cytometry-based quantification of MuSC numbers in adult and aged StemRep mice. n = 4 mice, 2 pools of 2 mice. (C) Quantification of MYF5-E2Crimson mean fluorescence intensity in MuSCs derived from adult and aged StemRep mice. (D and E) Proportion of MYF5^Low (D) and MYF5^High (E) MuSCs in adult and aged StemRep mice. n = 3 mice per group, each datapoint represents one mouse. (F–K) Representative images (F) and quantification of total PAX7 positive cells (G), proportion, and absolute numbers of MYF5^Low (H, J) and MYF5^High (I, K) cells on uninjured tibialis anterior cross-sections from adult and aged StemRep mice. n = 4 mice, each datapoint represents one mouse. Scale bar, 50 μm. Data presented are means ± SEM. ^∗p < 0.05, ^∗∗p < 0.01 in a two-tailed unpaired Student’s t test for each population (B, C, D, E, G, H, I, J, K).