Figure 4: Mesenchymal stem cells improve muscle regeneration by decreasing septic state and restoring affected mitochondrial parameters in satellite cells.

(a) Schematic representation of timing of sacrifice, post-caecal ligature puncture and injury (with notexin) plus MSCs grafting. (b) Haematoxylin and eosin staining of the TA 21 days (d) post sepsis and injury (n=16). Scale bar, 100 μm. (c) Haematoxylin and eosin of TA 21 days post sepsis and injury with injection 6 h post injury of mesenchymal stem cells (n=16). Scale bar, 100 μm. (d–f) Fractions of fibrotic area. (d) Fibrotic area of the muscle tissue after regeneration (21 days post injury) in control, septic and septic injected with MSC conditions. (e) Sirius Red staining of muscle tissue of septic mice 21 days post injury (n=16) Scale bar, 100 μm. (f) Sirius Red staining of muscle tissue 21 days post injury of septic mice treated with MSC injection (n=16). Scale bar, 100 μm. (g) Example of Luminex on interleukin-6 (IL-6) representing the level of protein expression in pg g⁻¹ of muscle tissue. Control are non-injured mice (n=4), septic Tg:Pax7nGFP at 24 h (n=4) and septic Tg:Pax7nGFP injected with MSCs (n=4). (h) TMRE label in injured (n=3); injured, septic TgPax7nGFP (n=3); and injured, septic, MSC-injected TgPax7nGFP (n=3) at 24 h. (i) ATP content relative to control (n=6), septic TgPax7nGFP (n=6) and septic TgPax7nGFP injected with MSCs (n=6) at 24 h. (j) Relative glycolytic and mitochondrial ATP content in controls (no injury no CLP), 24 h post sepsis, and septic Tg:Pax7nGFP injected with MSCs. (k) Quantification by RT–qPCR of PGC1a, Nox1, Hif1a, Sod1 and Sod2 expression. (l) Relative level of intensity of MitoTracker Deep Red in controls, septic Tg:Pax7nGFP and septic Tg:Pax7nGFP injected with MSCs at 24 h (n=6). (m) Quantification of mtDNA in FACS cell-sorted satellite cells in injured, injured and septic, and injured, septic and MSC-injected Tg:Pax7nGFP mice. (n) Agarose gel electrophoresis of long PCR amplification of mtDNA in SCs from controls, septic and septic mice injected with MSCs showing mtDNA alterations only in septic mice (left panel). The coordinates of the amplified fragments are shown in Fig. 3k. MSCs rescue normal mitochondrial genome size after sepsis. (o) Maximal tension of muscle fibres of septic and injured (n=6) compared with septic, injured and MSC-injected C57Bl/6 mice (n=7) according to the Ca²⁺ concentration. Data are represented as mean±s.d. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; not significant, compared with the respective control (Mann–Whitney test).