Neurogenic Effects of Cell-Free Extracts of Adipose Stem Cells

Abstract

Stem-cell-based therapies are regarded as promising treatments for neurological disorders, and adipose-derived stem cells (ASCs) are a feasible source of clinical application of stem cell. Recent studies have shown that stem cells have a therapeutic potential for use in the treatment of various illnesses through paracrine action. To examine the effects of cell components of ASCs on neural stem cells (NSCs), we treated cell-free extracts of ASCs (CFE-ASCs) containing various components with brain-derived NSCs. To elucidate the effects of CFE-ASCs in NSC proliferation, we treated mouse subventricular zone-derived cultured NSCs with various doses of CFE-ASCs. As a result, CFE-ASCs were found to induce the proliferation of NSCs under conditions of growth factor deprivation in a dose-dependent manner (p<0.01). CFE-ASCs increase the expression of neuron and astrocyte differentiation markers including Tuj-1 (p<0.05) and glial fibrillary acidic protein (p<0.01) without altering the cell's fate in differentiating NSCs. In addition, treatment with CFE-ASCs induces an increase in neurite numbers (p<0.01) and lengths of NSCs (p<0.05). Furthermore, CFE-ASCs rescue the hydrogen peroxide-induced reduction of NSCs' viability (p<0.05) and neurite branching (p<0.01). Findings from our study indicate that CFE-ASCs support the survival, proliferation and differentiation of NSCs accompanied with neurite outgrowth, suggesting that CFE-ASCs can modulate neurogenesis in the central nervous system.

Fig 1. Proliferation of NSC by CFE-ASCs.

Primary dissociated SVZ-derived NSC were maintained by the neurosphere method. SVZ tissue was isolated and digested from mice. NSCs were maintained in DMEM/F12/B27 with EGF and bFGF, forming neurospheres (A). After neurosphere cell expansion, these spheres were then transferred into growth-factor-free medium with 5% FBS and kept for 10 days. Without growth factors, spheres were dissociated and attached on coated cover glass (B). SVZ-derived NSCs were treated with CFE-ASCs and BrdU for 2 days in the absence of EGF and bFGF. BrdU (red) and DAPI (blue) staining was performed and observed with fluorescence microscope. Microscopic images showed that CFE-ASCs-treated NSCs have more BudU positive cells than vehicle-treated cells (C). BrdU positive cells were counted and normalized with positive DAPI. Relative cell numbers were represented as bar graphs (n = 5 per group) (D). Cell proliferation assays were performed and relative optical densities were represented as bar graphs (n = 4) (E). *p<0.01 compared with the control group (ANOVA followed by post-hoc test). All data are represented as the mean ± standard deviation (SD). Bar = 50 ìm.

Fig 2. Neural-differentiation promoting effects of CFE-ASCs.

NSCs were cultured with DMEM/F12 with B27 and 5% FBS without EGF and bFGF. After 6 days, NSCs were treated with CFE-ASCs or vehicle for 2 days and stained with Tuj-1, GFAP and DAPI. Fluorescence microscopic observation showed expression of Tuj-1 (Red) and GFAP (Green) (A). Positive-stained cells were counted and normalized with DAPI (Blue) count. Vehicle and CFE-ASC-treated NSCs showed no significant differences in positive-cell numbers (n = 5) (B). Fluorescence intensities of Tuj-1 and GFAP were calculated and normalized with DAPI. Relative fluorescence intensity was higher in the CFE-ASC-treated group compared with the vehicle group (n = 5) (C). The data were analyzed using Student’s t-test. All data are represented as the mean ± SD. *p<0.05; **p<0.01 compared with the control group. Bar = 100 ìm.

Fig 3. Induction of neurite genesis and growth by CFE-ASCs.

Optical microscopic observation was performed 2 days after CFE-ASC or vehicle treatment in differentiating NSCs. CFE-ASC-treated NSCs showed growth and genesis of neurites (A). The numbers and lengths of neurites were analyzed and represented as bar graphs (n = 8 per group) (B). The data were analyzed using Student’s t-test. All data represented as the mean ± SD. *p<0.05; **p<0.01 compared with the control group. Bar = 50 ìm.

Fig 4. Amelioration of oxidative stress in neural cells.

After attachment of NSCs for 7 days in the absence of EGF and bFGF, 10 mM hydrogen peroxide was added to medium with or without CFE-ASC for 2 days, and optical microscopic pictures were obtained (A). The survival rates of the attached neuronal cell population was obtained using WST-1 cell viability assay kits (B). The numbers of neurites were counted and represented as bar graphs. The data were analyzed using Student’s t-test. All data are represented as the mean ± SD. *p<0.05; **p<0.01 compared with the control group. Bar = 50 ìm.