Fig. 4

Homozygous Pax7-YFP mouse-derived satellite cells exhibit a wild-type stem cell function. a–c Regeneration was induced in the TA muscle of Pax7^+/+, Pax7^YFP/+, and Pax7^YFP/YFP mice by injection of CTX as shown in Fig. 3. Regenerating muscles were isolated at day 14 following CTX injection. Representative immunohistochemical images for Laminin α2 in cross-sections of TA muscle (a). Cross-sectional area (CSA) of centrally nucleated regenerating myofibers was quantified (b), scale bar 50 μm. (n = 4). Muscle weight of TA muscles was measured (c) (n = 3–7). Data represent means ± s.e.m. One-way ANOVA followed by the Bonferroni post hoc test: NS non-significant. d–j Satellite cell populations were isolated from limb muscles of Pax7^+/+ or Pax7^YFP/YFP mice by FACS and cultured as shown in Fig. 2. Time course for analysis (d). e Representative images of EdU staining of plated satellite cells in GM conditions [quantified in (f)]. Nuclei were stained with DAPI. > 300 cells per mouse were counted (n = 3 mice). Data represent means ± s.e.m. (NS non-significant), scale bar 50 μm. g Myogenic differentiation was induced under differentiation medium (DM) conditions for 4 days and cells immunostained for MyHC [differentiation index (h) was measured as the ratio of MyHC+ nuclei per total DAPI+ nuclei. > 200 cells per mouse were counted (n = 6–7 mice)], scale bar 20 μm. Data represent means ± s.d. (NS non-significant). i, j Self-renewal of satellite cells was induced under DM conditions for 4 days. (i) Co-immunostaining for PAX7 (or YFP) and MyoD. Arrows indicate Pax7⁺(or YFP+)/MyoD⁻ self-renewal cells [quantified in (j)], scale bar 10 μm. > 250 nuclei per mouse (n = 3 mice) were counted. Data represent means ± s.d. (NS non-significant). k–m To evaluate whether satellite cells isolated from Pax7^YFP/YFP mice were able to give rise to progeny and regenerate muscle, transplantation analysis was performed. k Time course for analysis of transplantation. YFP⁺ cells (5 × 10⁴ cells) were transplanted into CTX pre-injured TA muscle of mdx mice (n = 4). The contralateral legs were injected with PBS and used as a control. Muscles were removed for immunohistochemistry 14 days post-transplantation. l Immunohistochemistry for YFP and laminin α2 in cross-sections of TA muscles engrafted with YFP+ satellite cells. Arrows indicate YFP⁺ cells, scale bar 50 μm. m Immunohistochemistry of dystrophin and YFP proteins in TA muscle of mdx mice (n = 4). Pax7-YFP homozygous mouse-derived satellite cells were capable of producing self-renewed cells and restoring dystrophin in mdx mice. Arrows indicate YFP⁺ cells, scale bar 10 μm