Promoting potential of adipose derived stem cells on peripheral nerve regeneration

Abstract

The ultimate goal of treating peripheral nerve defects is reconstructing continuity of the nerve stumps to regain nerve conduction and functional recovery. Clinically, autologous nerve grafts and Schwann cell (SC) therapy have limitations, such as the need for secondary surgery, sacrifice of another nerve and donor site complication. Adipose derived stem cells (ADSCs) may promise to be ideal alternative cells of SCs. To explore the potential of ADSCs promoting peripheral nerve regeneration, the present study investigated the influences of ADSCs on proliferation and neurotrophic function of SCs using co‑culture model in vitro. Western blot analysis, immunocytochemistry, a cell viability assay, reverse transcription‑polymerase chain reaction (RT‑PCR) and ELISA were applied for examining the interaction of ADSCs and SCs in a co‑culture model in vitro. Western blot analysis and immunocytochemistry demonstrated that protein expression levels of glial filament acidic protein (GFAP) and S100 in ADSCs co‑cultured with SCs for 14 days were significantly higher compared with cells cultured alone. Cell viability assay indicated that the cell viability of SCs co‑cultured with ADSCs for 3, 4, 5, 6 and 7 days was significantly higher than those cultured alone. RT‑PCR showed that expression levels of neurotrophic factors [nerve growth factor (NGF) and glial cell line‑derived neurotrophic factor (GDNF)] and extracellular matrix components [fibronectin (FN) and laminin (LN)] in SCs co‑cultured with ADSCs for 14 days were significantly higher than those in SCs cultured alone. NGF, GDNF, FN and LN in the supernatants of co‑culture system were significantly higher than cells cultured alone, as ELISA revealed. The results of this study suggested that the transplantation of ADSCs may have a promoting potential to the peripheral nerve regeneration as undifferentiated state.

Figure 1.

Characteristics of rat ADSCs of 3 passages. (A) Rat ADSCs of 3 passages presented a large, flat and mono-layer shape, arranged in bundles or whorls. (B) Cell viability assay indicated that there was a lag phase of cell proliferation within 48 h, but expanded rapidly from 3 days and peaked at 6 days. (C) Flow cytometry demonstrated that rat ADSCs of passage 3 were CD29 and CD44 positive, but CD45 and CD11 negative. CD, cluster of differentiation; OD, optical density; ADSCs, adipose derived stem cells.

Figure 2.

Differentiation of rat ADSCs. When treated with appropriate lineage-specific induction medium, rat ADSCs of 3 passages were successfully differentiated into adipocytes and osteoblasts which were confirmed by (A) Oil-Red O staining and (B) Alizarin Red S staining. ADSCs, adipose derived stem cells.

Figure 3.

Neurotrophic secretion of rat ADSCs. (A) Immunocytochemistry indicated neurotrophic factors as NGF (panel a), GDNF (panel b), fibronectin (panel c) and laminin (panel d) could be detected in rat ADSCs and supernatants of cultured medium. Green fluorescence indicates the protein of interest, and blue fluorescence indicates nuclei. (B) ELISA results were consistent. Data are expressed as the mean ± standard deviation. *P<0.01 vs. control group. ADSCs, adipose derived stem cells.

Figure 4.

Differentiation of rat ADSCs into SCs-like cells. (A) 14 days after differentiation, rat ADSCs of 3 passages became smaller, had a spindle-like morphology, and were bipolar with long protrusions on two ends (magnification, ×100). Immunocytochemistry demonstrated that SC-like cells were positive for (B) S100 and (C) GFAP. Green fluorescence indicates the protein of interest, and blue fluorescence indicates nuclei. GFAP, glial filament acidic protein; SC, Schwann cell; ADSCs, adipose derived stem cells.

Figure 5.

Rat ADSCs in the co-culture system. (A) Rat ADSCs changed to smaller, spindle-like and bipolar morphology, with long protrusions on two ends after 14 days of co-culture (magnification, ×100). (B) Western blot analysis demonstrated that protein expression levels of GFAP and S100 in ADSCs co-cultured with SCs for 14 days were significantly higher than those in ADSCs cultured alone. (C) Immunocytochemistry revealed that S100 (panel a) and GFAP (panel b) were positive in SC-like cells and negative in the control group (panel c, panel d). GFAP, glial filament acidic protein; SC, Schwann cell; ADSCs, adipose derived stem cells.

Figure 6.

SCs in the co-culture system. (A) Cell viability assay indicated that the cell viability of SCs co-cultured with ADSCs for 3, 4, 5, 6 and 7 days was significantly higher than those cultured alone. (B) Secretion of neurotrophic factors in the co-culture system. NGF, GDNF, FN and LN in the supernatants of SCs in the co-culture system were significantly higher than in SCs cultured alone, as ELISA revealed. (C) Reverse transcription-polymerase chain reaction demonstrated that mRNA expression levels of neurotrophic factors (NGF, GDNF) and extracellular matrix components (FN, LN) in SCs co-cultured with ADSCs for 14 days were significantly higher than those in SCs cultured alone. Data are expressed as the mean ± standard deviation. *P<0.01 vs. control group. FN, fibronectin; LN, laminin; NGF, nerve growth factor; GDNF, glial cell line-derived neurotrophic factor; SC, Schwann cell; ADSCs, adipose derived stem cells; OD, optical density.