Therapeutic effect of human adipose-derived stem cells and their secretome in experimental diabetic pain

Abstract

Painful neuropathy is one of the complications of diabetes mellitus that adversely affects patients'quality of life. Pharmacological treatments are not fully satisfactory, and novel approaches needed. In a preclinical mouse model of diabetes the effect of both human mesenchymal stromal cells from adipose tissue (hASC) and their conditioned medium (hASC-CM) was evaluated. Diabetes was induced by streptozotocin. After neuropathic hypersensitivity was established, mice were intravenously injected with either 1 × 10⁶ hASC or with CM derived from 2 × 10⁶ hASC. Both hASC and CM (secretome) reversed mechanical, thermal allodynia and thermal hyperalgesia, with a rapid and long lasting effect, maintained up to 12 weeks after treatments. In nerves, dorsal root ganglia and spinal cord of neuropathic mice we determined high IL-1β, IL-6 and TNF-α and low IL-10 levels. Both treatments restored a correct pro/antinflammatory cytokine balance and prevented skin innervation loss. In spleens of streptozotocin-mice, both hASC and hASC-CM re-established Th1/Th2 balance that was shifted to Th1 during diabetes. Blood glucose levels were unaffected although diabetic animals regained weight, and kidney morphology was recovered by treatments. Our data show that hASC and hASC-CM treatments may be promising approaches for diabetic neuropathic pain, and suggest that cell effect is likely mediated by their secretome.

Figure 1

Experimental design. STZ: streptozotocin; hASC: human adipose-derived stem/stromal cells (1 × 10⁶ cells). hASC-CM: hASC-conditioned medium (from 2 × 10⁶ cells).

Figure 2

hASC and hASC-CM treatments reduce allodynia in STZ mice. (a–f) Effects of i.v. hASC (1 × 10⁶) or hASC-CM (obtained from 2 × 10⁶ cells) treatments on mechanical allodynia in STZ mice. (a,b) Mice received a single hASC or hASC-CM injection, 2 weeks after STZ, and the effects were monitored up to 14 weeks after STZ (long-lasting effects) (a) or few hours (3 to 72 h) after the injection (short-term effects) (b). (c,d) Mice received two repeated hASC or hASC-CM administrations 2 and 6 weeks after STZ. Long-lasting effects (c) and short-term effects after the second administration (d). (e,f) Mice received a single hASC or hASC-CM injection, 6 weeks after STZ. Long-lasting effects (e) and short-term effects (f). (g) Effect of hASC-CM and CM obtained from human fibroblasts (hF-CM), administered 2 weeks after STZ. Data represent mean ± SEM of 6–8 mice per group. Two-way ANOVA followed by Bonferroni’s test was used for multiple comparisons. ***p < 0.001 vs CTR; °°°p < 0.001 vs STZ; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs STZ + hASC; ^§§§p < 0.001 vs STZ + hASC-CM; ^£££p < 0.001 vs. W6. (h,i) effect of hASC and hASC-CM on thermal allodynia (h) and hyperalgesia (i). Treatments were performed 2 weeks after STZ and cold allodynia and hot plate thesholds were evaluated after 3, 24, 72 hours and 1 week after treatments. Values are mean ± SEM of 6 mice per group, and were compared with Mann–Whitney U-test (cold allodynia) and two-way ANOVA followed by Bonferroni’s test for multiple comparisons (thermal hyperalgesia). *p < 0.05, ***p < 0.001 vs CTR; °p < 0.05, °°°p < 0.001 vs STZ.

Figure 3

hASC tracking by human ALU sequence detection. Summary of the Alu PCR products observed in lung, liver, pancreas and sciatic nerve after 1 and 3 days and 1, 2 and 3 weeks from hASC administration (a). Representative gels showing human ALU sequence detection in STZ and naïve (CTR) mice tissues collected 1 week (W1) and 2 weeks (W2) after treatment (b). Lu: lung; Li: liver; P: pancreas; N: sciatic nerve; + : positive control; −: negative control.

Figure 4

hASC and hASC-CM maintain a correct pro- and anti-inflammatory cytokine balance in sciatic nerves, DRG and spinal cord of STZ mice. IL-1β, IL-6, TNFα and IL-10 protein content in nervous tissues was evaluated by ELISA and reported as pg cytokine/mg total protein. (a–d) IL-1β (a) IL-6 (b), TNF-α (c) and IL-10 (d) in sciatic nerve, DRG and spinal cord of STZ mice treated 2 weeks after STZ with hASC or hASC-CM; cytokines were evaluated after 1 week from treatments. (e,f) IL-1β (e) and IL-10 (f) levels in spinal cord, measured 14 weeks after STZ in animals treated with hASC or hASC-CM either 2 weeks (W2) or 6 weeks (W6) after STZ. Data represent mean ± SEM of 6 mice per group. One-way ANOVA was used for statistical evaluation, followed by Bonferroni’s post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001 vs CTR; °p < 0.05, °°p < 0.01, °°°p < 0.001 vs STZ; ^###p < 0.001 vs STZ + hASC.

Figure 5

hASC and hASC-CM prevent alteration of DRG CGRP, thickness reduction and nerve fiber loss in paw skin of STZ mice. (a) DRG and spinal cord levels of CGRP measured 3 weeks after STZ in diabetic mice treated with hASC or hASC-CM 2 weeks after STZ. Data represent mean ± SEM of 6 mice per group. One-way ANOVA was used for statistical evaluation, followed by Bonferroni’s post hoc test for multiple comparisons. ***p < 0.001 vs CTR; °°p < 0.01 vs STZ. Microphotographs of plantar skin after PGP9.5 + immunohistochemistry and counterstaining with haematoxylin at 3 (b) and 14 weeks (e) after STZ, in CTR, STZ, STZ + hASC and STZ + hASC-CM groups. Animals were treated 2 weeks after STZ. Nerve fibers are stained in brown; arrow heads indicate PGP9.5⁺ fibers. Quantitative evaluation of epidermal thickness at 3 weeks (c) and 14 weeks (f) and of PGP9.5 immunopositivity as percentage of immunopositive area in epidermal and subepidermal area at 3 (d) and 14 (g) weeks after STZ. Data represent mean ± SEM and were compared by One-way ANOVA followed by a Bonferroni’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 vs CTR; °p < 0.05, °°°p < 0.001 vs STZ; ^###p < 0.001 vs STZ + hASC.

Figure 6

Effect of hASC and hASC-CM on body weight and blood glucose levels in STZ mice. (a,b) Body weight of STZ mice treated with hASC or hASC-CM 2 weeks (a) and 6 weeks (b) after STZ. (c,d) Blood glucose levels of STZ mice treated with hASC or hASC-CM 2 weeks (c) and 6 weeks (d) after STZ. Data are means ± SD of 6/8 animals. Two-way ANOVA was used for statistical evaluation, followed by Bonferroni’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001 vs CTR; °p < 0.05, °°p < 0.01, °°°p < 0.001 vs STZ.

Figure 7

hASC and hASC-CM treatments modulate cytokine release from splenocytes. IFN-γ (a and b), IL-2 (c and d), IL-4 (e and f) and IL-10 (g and h) were evaluated by ELISA, and reported as protein concentrations in culture media. (a,c,e and g) depict the levels of cytokine evaluated 3 weeks after STZ and 1 week after hASC or hASC-CM treatments. (b,d,f and h) Report cytokines levels measured 14 weeks after STZ in animals treated with hASC or hASC-CM either 2 weeks (W2) or 6 weeks (W6) after STZ. Data represent mean ± SEM of 6 mice per group, and have been statistically analyzed with One-way ANOVA, followed by Bonferroni’s test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001 vs CTR; °p < 0.05, °°p < 0.01, °°°p < 0.001 vs STZ.