Neurogenic Effects of Cell-Free Extracts of Adipose Stem Cells

<div><p>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.</p></div

Induction of neurite genesis and growth by CFE-ASCs.

<p>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.</p

Proliferation of NSC by CFE-ASCs.

<p>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.</p

Amelioration of oxidative stress in neural cells.

<p>After attachment of NSCs for 7 days in the absence of EGF and <u>bFGF</u>, 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.</p

Neural-differentiation promoting effects of CFE-ASCs.

<p>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.</p

Amelioration of Huntington's disease phenotypes by Beta-Lapachone is associated with increases in Sirt1 expression, CREB phosphorylation and PGC-1α deacetylation

<div><p>Huntington’s disease (HD) is one of the most devastating genetic neurodegenerative disorders with no effective medical therapy. β-Lapachone (βL) is a natural compound obtained from the bark of the Lapacho tree and has been reported to have beneficial effects on various diseases. Sirt1 is a deacetylase of the sirtuin family and deacetylates proteins including the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) which is associated with mitochondrial respiration and biogenesis. To examine the effectiveness of βL on HD, βL was orally applied to R6/2 HD mice and behavioral phenotypes associated with HD, such as impairment of rota-rod performance and increase of clasping behavior, as well as changes of Sirt1 expression, CREB phosphorylation and PGC-1α deacetylation were examined. Western blot results showed that Sirt1 and p-CREB levels were significantly increased in the brains of βL-treated R6/2 mice. An increase in deacetylation of PGC-1α, which is thought to increase its activity, was observed by oral administration of βL. In an <i>in vitro</i> HD model, βL treatment resulted in an attenuation of MitoSOX red fluorescence intensity, indicating an amelioration of mitochondrial reactive oxygen species by βL. Furthermore, improvements in the rota-rod performance and clasping score were observed in R6/2 HD mice after oral administration of βL compared to that of vehicle control-treated mice. Taken together, our data show that βL is a potential therapeutic candidate for the treatment of HD-associated phenotypes, and increases in Sirt1 level, CREB phosphorylation and PGC-103B1 deacetylation can be the possible underlying mechanism of the effects of βL.</p></div

Reduction in PGC-1α acetylation by βL administration in mice.

<p>βL was orally administered to R6/2 mice for 6 weeks from 5 to 11 weeks of age. After the sacrifice, brains were isolated and an immunoprecipitation was performed using the PGC-1α antibody. Immunoprecipitated proteins were separated by SDS-PAGE and blotted with anti-PGC-1α and anti-acetylated-lysine antibodies to measure the level of PGC-1α acetylation. Relative band intensities were analyzed using the Image J software. The acetylation level of PGC-1α was analyzed by the intensity of acetylated lysine relative to the amount of total PGC-1α (lower panel). The graph shows means ± SEM. *p < 0.01 indicates significant differences when compared to the control group.</p

Decrease in mitochondrial superoxide level by βL treatment of an <i>in vitro</i> HD model.

<p>R6/2 mouse-derived NSCs were cultured and differentiated in a differentiation medium. After differentiation, βL was added for 3 days and mitochondrial superoxide levels were measured by immunocytochemistry (A) and flow cytometry (B) after MitoSOX red staining. The red, roundish objects are individual cells with MitoSOX staining, and the M1 marker of the flow cytometry result indicats a Cy3-positive population. The percentage values of NSCs with M1 after vehicle or βL treatment are represented in a bar graph (n = 3) (C). The graph shows means ± SEM. *p < 0.05 indicates significant differences when compared to the vehicle-treated group.</p

Cytosolic Extract of Human Adipose Stem Cells Reverses the Amyloid Beta-Induced Mitochondrial Apoptosis via P53/Foxo3a Pathway

<div><p>Human adipose stem cells (hASC) have therapeutic potential for the treatment of neurodegenerative disorders. Mitochondrial dysfunction is frequently observed in most neurodegenerative disorders, including Alzheimer’s disease. We explored the therapeutic potential of hASC cytosolic extracts to attenuate neuronal death induced by mitochondrial dysfunction in an Alzheimer’s disease (AD) <i>in vitro</i> models. Amyloid beta (Aβ) was used to induce cytotoxity in an immortal hippocampal cell line (HT22) and neuronal stem cells from the brain of TG2576 transgenic mice were also used to test the protective role of hASC cytosolic extracts. Cell viability and flow cytometry results demonstrated that the hASC extract prevents the toxicity and apoptosis in AD <i>in vitro</i> models. Moreover, JC-1 and MitoSoxRed staining followed by fluorescence microscopy and flow cytometry results showed that the hASC extract ameliorated the effect of Aβ-induced mitochondrial oxidative stress and reduced the mitochondrial membrane potential. Western blot result showed that hASC extract modulated mitochondria-associated proteins, such as Bax and Bcl2, and down-regulated cleaved caspase-3. In addition, hASC extract decreased Aβ generation and reversed up-regulated p53 and foxo3a protein level in AD <i>in vitro</i> model cell derived from TG2576 mice. Taken together, these findings implicate a protective role of the hASC extract in the Aβ-induced mitochondrial apoptosis via regulation of P53/foxo3a pathway, providing insight into the molecular mechanisms of hASC extract and a therapeutic strategy to ameliorate neuronal death induced by Aβ.</p></div

The hASC extract prevents Aβ-induced cell toxicity.

<p>HT22 cells were treated with 100 μg/ml Aβ with or without 30 μg/ml of the hASC extract. At 48 h, (A) cells were directly imaged with a microscope. Representative images are shown. (B) The cell viability assay using CCK8 shows the reduction of cell viability by treatment with Aβ and the hASC extract; normalized values are presented (<i>n</i> = 3 each). (C, D) Cells were subjected to Annexin V-FITC and (or) PI staining. Images captured by a fluorescent microscope; or cells were analyzed using flow cytometry and quantified graph shows the level of apoptosis in the experiments (<i>n</i> = 3 each). Scale bar = 10 μm (A); 50 μm (C). Error bars represent S.E.M. *p<0.05, **p<0.01.</p