Therapeutic potential of adipose-derived stem cells and macrophages for ischemic skeletal muscle repair

Abstract

Aim: Progressive ischemia due to peripheral artery disease causes muscle damage and reduced strength of the lower extremities. Autologous cell therapy is an attractive treatment to restore perfusion and improve muscle function. Adipose-derived stem cells (ASCs) have therapeutic potential in tissue repair, including polarizing effects on macrophages (MPs).

Materials & methods: Co-culture systems of ASCs and MPs were analyzed for gene and protein expression modifications in ASC-conditioned MPs. Co-transplantation of MPs/ASCs in vivo led to improved skeletal muscle regeneration in a mouse model of peripheral artery disease.

Results: ASCs/MPs therapy restored muscle function, increased perfusion and reduced inflammatory infiltrate.

Conclusion: Combined MPs/ASCs cell therapy is a promising approach to restore muscle function and stimulate local angiogenesis in the ischemic limb.

Keywords: adipose-derived stem cells; cell-mediated therapy; ischemic injury; macrophages; peripheral artery disease; skeletal muscle regeneration.

Figure 1.. Hypoxia-induced changes in individual human adipose-derived stem cells and U937 macrophages secretomes, and adipose-derived stem cells-induced alterations of functional macrophage phenotype in co-cultures.

(A) Modified secretion of soluble factors by hASCs cultured under 1–2% oxygen for 48 h. The hASCs conditioned media was collected following 1-2% oxygen hypoxic cultivation and analyzed using an ELISA-based array kit. Representative blotted membranes are shown for hASCs on the left. The heat maps were colorized based on the percentage change relative to normoxia controls. The data shown were semi-quantitatively derived from chemiluminescent images based on the signal intensity. (B) U937 human macrophages were cultured in the bottom of a transwell insert under normoxia and hypoxia, alone or in the presence of hASCs. Values are displayed as mean ± standard deviation. *p < 0.05 compared with normoxic condition. hASC: Human adipose-dervied stem cell.

Figure 2.. Gene-expression profile and CD206 cell surface marker expression of in vitro polarized bone marrow macrophages.

(A) Bone marrow MPs were cultured with either IFN-γ (20 ng/ml) or IL-4 (20 ng/ml) for 48 h. MPs maintained in M-CSF were used as controls (=1). RNA was isolated and gene expression was quantified using SYBR real-time polyamerase chain reaction and double delta Ct method; *p < 0.05, Student's t-test. (B) Flow cytometry was used to characterize the expression of mannose receptor (CD206) on the surface of bone-marrow derived MPs after in vitro polarization and following direct co-culture with mASCs. Values are displayed as mean ± standard deviation. *p < 0.05 relative to M-CSF treated MPs (control). ^#p < 0.05 relative to IFN-γ polarized MPs. ^&p < 0.05 relative to IL-4 polarized MPs. ASC: Adipose-derived stem cell; MP: Macrophage; M-CSF: Macrophage colony-stimulating factor.

Figure 3.. Direct correlation between the number of mouse adipose-derived stem cells and expression levels of mannose receptor on the surface of mouse bone marrow macrophages in direct co-cultures.

Flow cytometry was used to characterize the expression of mannose receptor (CD206) after varying percent mouse ASCs (mASC) in co-cultures from 0 (0:1; mASC:mouse bone marrow MPs [mMPs]) to 20% (1:5; mASC/mMPs). Strong correlation exists between percent (%) mASCs in direct co-culture and subsequent percent (%) MPs expressing CD206. ASC: Adipose-derived stem cell; CC: Co-culture; MP: Macrophage.

Figure 4.. Gene-expression profile and CD206 surface expression of mouse bone marrow macrophages after direct co-culture with mouse adipose-derived stem cells under normoxic and hypoxic conditions.

(A) Mouse bone marrow MPs (mMPs) were co-cultured with mouse ASCs (mASCs) under normoxia (20% oxygen) for 48 h. MPs cultured alone were used as controls (=1). Expression levels of mannose receptor (CD206) were confirmed by flow cytometry. RNA was isolated and gene expression was quantified using SYBR real-time polyamerase chain reaction. (B) mMPs were co-cultured with mASCs under hypoxia (1–2% oxygen) for 48 h. MPs cultured alone were used as controls (=1). Values are displayed as mean ± standard deviation. *p < 0.05 Student's t-test. ASC: Adipose-derived stem cells; MP: Macrophage.

Figure 5.. Co-delivery of mouse bone marrow macrophages/mouse adipose-derived stem cells after femoral artery excision injury leads to improved muscle histopathology and contractile function at 3 weeks of recovery.

(A) Representative images of hematoxylin and eosin staining (20X; scale bar: 100 μm) and quantification of average myofiber sizes in contralateral controls and treatment groups; (B) Characterization of muscle contractile properties by evaluating muscle mass (mg), tetanic tension (N) and normalized force (N/mg). n = 5–9 for each group; three images/animal. Values are expressed as mean ± standard deviation. *p < 0.05 compared to contralateral side. ^#p < 0.05 compared with M0 group. ⁺p < 0.05 compared with PBS group using one-way ANOVA with post hoc Tukey honest signicant difference. ASC: Adipose-derived stem cell; MP: Macrophage; PBS: Phosphate-buffered saline.

Figure 6.. Skeletal muscle angiogenesis is significantly improved 3 weeks after mouse bone marrow macrophages/mouse adipose-derived stem cell co-delivery into femoral artery excision injured muscles.

(A) Representative images of laser speckle and (B) CD31 staining of ischemic limbs (20X), scale bar 100 μm. Quantification of (C) blood flow relative to contralateral, uninjured side. (D) Capillary per myofiber ratio. n = 3–5 for each group; three images/animal. Values are expressed as mean ± standard deviation. ^&p < 0.05 compared to D1 post-femoral artery excision using one-way analysis of variance with post hoc Dunnett's test. *p < 0.05 compared to contralateral side. ^#p < 0.05 compared with M0 group. ⁺p < 0.05 compared with PBS group using one-way analysis of variance with post-hoc Tukey honest significant difference. ASC: Adipose-derived stem cell; FAE: Femoral artery excision; MP: Macrophage; PBS: Phosphate-buffered saline.

Figure 7.. Therapeutic transplantation of mouse bone marrow macrophages/mouse adipose-derived stem cell exhibit immunomodulatory function at 3 weeks after treatment and increases MyoD expression 4 days post-femoral artery excision.

Therapeutic transplantation of mMPs/mASCs exhibits immunomodulatory function at 3 weeks after treatment and increases MyoD expression 4 days post-femoral artery excision. (A) Representative images of CD45-specific staining (20X) in brown, scale bar: 100 μm. (B) Quantification of the number of inflammatory cells in ischemic muscles per field of view 3 weeks post-femoral artery excision, n = 5 for each group; 3 images/animal. (C) Representative images of MyoD staining (40X) in injured skeletal muscle (MPs/ASCs group), scale bar: 50μm. (D) Quantification of percent of MyoD+DAPI+ myonuclei out of total DAPI+ cells, n = 3 for each group; 5–7 images/animal. Values are expressed as mean ± standard deviation. ^#p < 0.05 compared with PBS group. ⁺p < 0.05 compared with mouse bone marrow MPs group using one-way analysis of variance with post hoc Tukey honest significant difference. ASC: Adipose-derived stem cell; MP: Macrophage; PBS: Phosphate-buffered saline.