Figure 5. Ex vivo-expanded aged MuSC progeny reconstitute the stem cell reserve in vivo and serially transplant.

(a, b, d–f) Analyses of stem cell repopulation in primary recipients and transplant engraftment in secondary recipients comparing 200 freshly isolated aged GFP/Luciferase MuSCs and the progeny of 200 aged MuSCs after culture on hydrogel ± SB202190 (SB; 10 µM). (a) Serial transplant scheme. (b) Frequency of donor-derived (GFP⁺) cells in the CD34⁺α7-integrin⁺ MuSC population in primary recipient muscles (n = 8–10 per condition). Box-and-whisker plot with median line. (c) Immunohistochemical (IHC) analysis of representative transverse section from notexin-injured muscles transplanted with the progeny of 100 aged Myf5^nLacZ/+ MuSCs after culture on hydrogel with 10 µM SB. Arrowhead indicates Myf5–β-gal⁺ cell in satellite position. Scale bar, 50 µm. (d–f) Engraftment analysis of secondary transplant recipients (n = 4). (d) BLI signals for up to 2 months after transplant (mean ± s.e.m.). Dashed line represents engraftment detection threshold. p, photons. (e) Representative BLI images at 4 weeks. Engraftment ratios are noted. (f) GFP immunohistochemistry (IHC) of transverse section from representative secondary recipient of SB/hydrogel aged MuSC progeny primary transplant. Scale bar, 75 µm. P < 0.05 (*) or P < 0.001 (***) by unpaired t test in (b) or Fisher’s exact test in (e).