Stem cell secretome treatment improves whole-body metabolism, reduces adiposity, and promotes skeletal muscle function in aged mice

Abstract

Aging coincides with the progressive loss of muscle mass and strength, increased adiposity, and diminished physical function. Accordingly, interventions aimed at improving muscle, metabolic, and/or physical health are of interest to mitigate the adverse effects of aging. In this study, we tested a stem cell secretome product, which contains extracellular vesicles and growth, cytoskeletal remodeling, and immunomodulatory factors. We examined the effects of 4 weeks of 2×/week unilateral intramuscular secretome injections (quadriceps) in ambulatory aged male C57BL/6 mice (22-24 months) compared to saline-injected aged-matched controls. Secretome delivery substantially increased whole-body lean mass and decreased fat mass, corresponding to higher myofiber cross-sectional area and smaller adipocyte size, respectively. Secretome-treated mice also had greater whole-body physical function (grip strength and rotarod performance) and had higher energy expenditure and physical activity levels compared to control mice. Furthermore, secretome-treated mice had greater skeletal muscle Pax7+ cell abundance, capillary density, collagen IV turnover, reduced intramuscular lipids, and greater Akt and hormone sensitive lipase phosphorylation in adipose tissue. Finally, secretome treatment in vitro directly enhanced muscle cell growth and IL-6 production, and in adipocytes, it reduced lipid content and improved insulin sensitivity. Moreover, indirect treatment with secretome-treated myotube culture media also enhanced muscle cell growth and adipocyte size reduction. Together, these data suggest that intramuscular treatment with a stem cell secretome improves whole-body metabolism, physical function, and remodels skeletal muscle and adipose tissue in aged mice.

Keywords: adipogenesis; fibrosis; lipids; metabolic rate; sarcopenia; stem cells.

FIGURE 1

Whole‐Body Metabolic Profile. Oxygen consumption (VO₂ – ml/kg/hr) and carbon dioxide production (VCO₂ – ml/kg/hr) across 24 h in comprehensive lab animal monitoring system (CLAMS) for control and secretome‐treated mice (a, b). 24‐h average VO₂ (c) and VCO₂ (d), 12‐h average dark cycle respiratory exchange ratio (RER – VCO₂/VO₂; e), 24‐h average energy expenditure (f; kcal/kg/hr), activity (g; movement – X + Y + Z), and food intake (g) (h). All data presented as mean ± SD, white bars represent controls while blue squares represent secretome‐treated mice. n = 6 for both groups. * indicates significant difference between conditions for the indicated timepoints with * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

FIGURE 2

Whole body Tissue and Physical Function Changes. Weekly changes lean mass (a), fat mass (b), body fat % (c), and body mass (d) for control and secretome‐treated mice. Weekly whole‐body grip strength (e) and change (Δ) in rotarod performance time from baseline (f). Quadriceps mass (g) and fat mass (h) following 4‐weeks. Control mice are represented by black circles and bars while secretome‐treated mice are noted by blue squares and bars. All data presented as mean ± SEM. (a–h) control mice (n = 15), secretome‐treated mice (n = 16). Weekly changes analyzed via mixed‐effects model with Holm‐Bonferroni multiple comparisons plus planned comparison t‐tests at the 4‐week timepoint. # indicates significant difference (p < 0.05) from baseline for respective group. * indicates significant difference (p < 0.05) between groups at the indicated time point.

FIGURE 3

Skeletal Muscle Morphology. Average quadriceps fiber cross sectional area μm² (a), minimum feret diameter μm² (b), and fiber type proportion (c; MyHC – IIa/IIb %) for control (n = 8) and secretome treated (n = 8) mice. Size distribution (%) for total (e), IIa (f) and IIb (g) fiber types across 500 μm² increments. Representative histochemical image of fiber type including cell border with laminin in blue, MyHC IIa in green, MyHC IIb in red, and scale bar of 50 μm (d). All data presented as mean ± SD, white circles and bars represent controls while blue squares and bars represent secretome‐treated mice. n = 8 for each group. Analyzed via t‐tests and mixed effects models with Holm‐Bonferroni comparisons. * indicates significant difference between groups at indicated category with * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

FIGURE 4

Skeletal Muscle Remodeling. Muscle satellite cell content (Pax7⁺/DAPI⁺ − co‐localization) corrected to number of fibers (a). Representative image of satellite cell localization with PAX7⁺ in off‐red/pink, laminin fiber borders in green, DAPI in blue, satellite cells indicate by white arrows, and scale bar of 50 μm² (b). Fiber capillarization (CD31⁺) corrected to number of muscle fibers (c). Representative image of capillarization with CD31⁺ in red, laminin in green, and DAPI in blue (d). Ratio of B‐CHP to COL‐IV (e) and representative image with B‐CHP in purple and COL‐IV in yellow (f). All data presented as mean ± SD, white circles and bars represent controls while blue squares and bars represent secretome‐treated mice. Control mice (n = 8), secretome‐treated mice (n = 7–8), right (injected) quadriceps assessed. Analyzed via t‐tests. * indicates significant difference between conditions for the indicated timepoints with * = p < 0.05, ** = p < 0.01.

FIGURE 5

Adipose Morphology and Muscle Lipid Content. Average cellular diameter μm of I‐WAT (a) and E‐WAT (b) depots. Size distribution (%) of I‐WAT (c) and E‐WAT (d) cells across 5 μm increments. Average liver lipid droplet (e) and fibrosis (f; trichrome staining) area (%) as assessed with H&E. Representative image of I‐WAT and E‐WAT depots as well as liver H&E and trichrome staining with scale bar 50 μm (g). Protein phosphorylation status for protein kinase B (Akt) and hormone sensitive lipase (HSL) for I‐WAT and E‐WAT depots and representative western blot image (h). Muscle lipid content including total triglycerides (TAGs) diglycerides (DAGs), ceramides (Cer), and C18:0 ceramide (C18:0 Cer). All data presented as mean ± SD, white circles and bars represent controls while blue squares and bars represent secretome treated groups. (a–g) n = 8 for each group, (h–i) n = 7 for control, n = 6 for secretome, (i) n = 6 for control, n = 8 for secretome. Analyzed via t‐tests (a, b, e, f, h, i) or two‐way ANOVA with Holm‐Bonferroni comparison (c, d). * indicates significant difference between groups at indicated category with * = p < 0.05, ** = p < 0.01.

FIGURE 6

Direct and Indirect Cellular Experiments. Cell culture design for media replacement and cultured media (CM) experiments (a). Average myotube area (%) (b) and myonuclear fusion index (au) (c) for control, secretome treated, control CM, and secretome CM conditions in differentiated C2C12 myotubes. Interleukin 6 (IL‐6) content in the culture media collected from secretome treated (4%) and control C2C12 myotubes (Fold change) (d). Average lipid droplet area corrected to DAPI area (Fold Change) in 3T3‐L1 adipocytes for control, 5 and 20% secretome product media replacement (e). Phosphorylated corrected to total Akt protein (fold change) for 20% secretome treated and control 3T3‐L1 adipocytes following overnight fast and insulin (100 nM) stimulation (f). Average lipid droplet area corrected to DAPI area (Fold Change) in 3T3‐L1 adipocytes for control and 20% media replacement with culture media from control and secretome treated C2C12 cells (g). Representative images of myotubes (h) and adipocytes treated with secretome (i) and culture media (j). (b–g) n = 4–7 per group or replicate. Analyzed via one‐way ANOVA with Holm‐Bonferroni comparison (b–c, e, g) or t‐tests (d, f). * indicates significant difference between groups as indicated with * = p < 0.05, ** = p < 0.01, *** = p < 0.001.