The heterozygous AβPPswe/PS1dE9 double-transgenic mouse with C57BL/6 background was used in this study. The mouse harbors the mutant human genes APPswe (Swedish mutations K594N/M595L) and presenilin-1 with the exon-9 deletion (PS1-dE9) under the control of mouse prion protein promoter. This type of transgenic mouse has been used widely in the study of AD [16, 17]. The mouse shows typical characteristics of AD and exhibits an early appearance of amyloid plaques deposition and increased proinflammatory microglial activation [17, 18]. Because of the gender-specific differences in the progression of amyloid plaque deposition, we used only male mice in this study. The mice were obtained from Beijing HFK Bio-Technology Co., Ltd., Institute of Laboratory Animal Science, Chinese Academy of Medical Science (Beijing, China), and housed in temperature- and humidity-controlled rooms on a 12 hour/12 hour light/dark cycle. Breeder mice carrying the transgenes are backcrossed to the C57BL/6 strain for six to seven generations to obtain mice used in research. The mice are genotyped to confirm the presence of the mutant genes by polymerase chain reaction (PCR) amplification of genomic DNA extracted from tail clippings before the mice are sold to research laboratories. All the procedures described in this study were in accordance with the Ethical Committee for Animal Experiments of Shandong University.
HUMSC isolation and in vitro culture
Human umbilical cords were obtained from full-term deliveries with the informed consent from parents after caesarian section. The procedure for collecting tissues was approved by the ethical committee of the Second Hospital of Shandong University. The procedure was based on the previous description by Huang et al.. After arteries and veins were removed, the remaining tissue, Wharton jelly, was cut into 0.5- to 1-cm3 pieces and suspended in Dulbecco modified Eagle low-glucose media (DMEM-LG; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco), 100 mg/ml penicillin, and 100 mg/ml streptomycin. The tissue was left undisturbed for 7 days in a 37°C humidified incubator with 5% CO2 to allow cells to migrate from the explants. Culture media was replaced every 3 days. The cells were passaged by using 0.25% trypsin-EDTA solution when they reached 80% to 90% confluence. The cells were analyzed with flow cytometry to confirm the immune-phenotype of HUMSCs according to the previous report . The cells used in this study were positive for CD73, CD90, and CD105, but negative for CD34, CD45, and HLA-DR, consistent with HUMSC characteristics.
Induction of neuron-like cells from HUMSCs
After HUMSCs were passaged 2 to 6 times and reached subconfluence, the cells were washed twice with basal DMEM media (without FBS) and divided into two groups. In the control group, the cells were cultured in basal DMEM medium (without FBS); in the D609 treatment group, the cells were cultured in basal media containing 60 μg/ml D609 (J&K, Beijing, China) to induce neuronal differentiation. D609 was prepared freshly in deionized water. We determined the optimal working concentration of D609 by performing a dose response of HUMSCs to 2 to 100 μg/ml of D609, and we found that 60 μg/ml D609 was the optimal concentration for neuronal differentiation (data not shown). The morphology of cells was observed under a phase-contrast microscope.
To determine the expression of neuronal cell markers, HUMSCs grown on glass coverslips were treated with D609 or basal medium for 6 hours. The cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes and then washed 3 times with PBS. The cells were blocked with normal goat serum for 20 minutes at room temperature and then incubated in the following primary antibodies: neuron-specific enolase (NSE, rabbit IgG, 1:200, Abcam, Cambridge, MA, USA), microtubule-associated protein 2 (MAP2, rabbit IgG, 1:200, Abcam), and glial fibrillary acidic protein (GFAP; rabbit IgG, 1:200, Sigma, St Louis, MO, USA). The cells were then washed in PBS and incubated with the secondary antibodies (goat anti-rabbit IgG-TRITC) for 1 hour at room temperature. After washing with PBS, the cells were observed under a fluorescence microscope (Olympus 1 × 71S1F-3, Tokyo, Japan). The differentiation rate of HUMSCs was calculated according to the following formula: The differentiation rate (percentage) = (the number of NSE or MAP2 positive cells/the total number of cells) ×100. For each sample, 10 randomly selected visual fields were used for the calculation. The results presented are the mean ± SEM from three independent experiments.
Transplantation of HUMSC-NCs in AβPP/PS1 double-transgenic mice
HUMSC-NC suspension, HUMSC suspension, or PBS was injected into 6-month-old male AβPP/PS1 transgenic mice for only one time. Before the injection, the cell suspension was washed with PBS for 3 times to remove drug and serum. Animals were anesthetized with 10% chloral hydrate and placed in a small animal stereotaxic apparatus with a microinjector unit. Either cell suspension or PBS was injected into mouse bilateral hippocampus at the following coordinates: 1.6 mm posterior to the bregma, 1.7 mm bilateral to the midline, and 2.5 mm ventral to the skull surface. Cell suspension was injected with a 25-μl syringe. Then 2 μl of cell suspension (approximately 5 × 104 cells) was injected into the hippocampus bilaterally. Cell suspension or PBS was delivered at a rate of 0.3 μl/min. After injection, the needle was left in place for 5 minutes before being retracted.
Three weeks after transplantation, we used the modified Morris water-maze test to assess spatial memory performance . The procedure consisted of 1 day of adapting tests without platform and 5 days of hidden-platform tests, plus a spatial probe test 24 hours after the last hidden-platform test; 15 mice were used in each group. The detailed method was described in our previous report . In brief, in the spatial-acquisition tests, mice were released into the pool and given 60 seconds to find the hidden platform. If a mouse did not find the platform within 60 seconds, it was guided to the platform. Animals were given four trials per day. The distal starting positions were semirandomly selected. For basic acquisition training, the platform was located in the southwest quadrant. The starting positions were north, east, southeast, and northwest. The time to find the platform was recorded as the latency for each trial. A single probe trial, in which the platform was removed, was performed after the hidden-platform task had been completed. Mice were placed in a novel start position (northeast) in the maze, and each mouse was allowed to swim for 60 seconds. The data were analyzed with multivariate analysis of variance (ANOVA). Mouse behavior was observed blindly. Observers were kept ignorant of the treatment given to each animal group.
Thioflavin S staining and immunohistochemical staining
AβPP/PS1 mice were killed after the behavior test. The mice were anesthetized with chloral hydrate, and then were immediately cardiac perfused with 0.9% saline solution followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). After the perfusion, the brains of the mice were excised and postfixed overnight at 4°C. The brain tissue was then incubated in 30% sucrose at 4°C until equilibration (six mice per group). Then 30- or 10-μm coronal sections were cut with a freezing microtome (Leica CM1850, Leica Microsystems, Heidelberg Germany) and stored at −20°C.
Thioflavin S staining was performed on floating sections (30-μm thickness). Brain sections were incubated in 0.5% thioflavin S (Sigma-Aldrich, USA) dissolved in 50% ethanol for 5 minutes, and then washed twice with 50% ethanol for 5 minutes each time. The brain sections were washed once with tap water for 5 minutes, and then mounted with mounting medium . The green fluorescence-stained plaques were observed under a fluorescence microscope. Frontal cortex, cingulate, and hippocampus were examined for amyloid load. According to the previous report , these regions have plaque prevalence in AD patients and are involved in memory functions.
For immunohistochemical staining, frontal cortex and hippocampus sections (10-μm thickness) were incubated with primary antibody at 4°C overnight. The following primary antibodies were used: Iba-1 (rabbit IgG, 1:500, Wako, Richmond, VA, USA); IL-4 (goat IgG, 1:200, Santa Cruz, Dallas, Texas, USA); TNF-α (goat IgG, 1:200, Santa Cruz); AMCase, (goat IgG, 1:200, Santa Cruz). Fluorescent dye-conjugated secondary antibodies (IgG-FITC or IgG-TRITC) were used to visualize the staining. To quantify the IHC staining, 10 serial cortex and hippocampal sections (at an interval of 50 μm for each section) from each animal (n = 6 for each group) were used to quantify each parameter. The staining was analyzed with the image-analyzing system, Image Pro Plus 6 (Media Cybernetics, Rockville, MD, USA).
Western blot analysis
Cerebral cortex and hippocampus from one hemisphere were isolated from AβPP/PS1 mice of each group after the behavior test (six mice per group). Brain tissue samples were snap frozen and stored at −80°C for future experiment. The brain samples were homogenized in ice-cold RIPA lysing buffer (Beyotime, Shanghai, China). The homogenized samples were centrifuged at 12,000 g for 20 minutes at 4°C. The supernatant was collected for Western blot. The proteins were separated with SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk in TBST for 1 hour and incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Synapsin I (rabbit IgG, 1:1,000, Abcam), NEP (goat IgG, 0.2 μg/ml, R&D, Minneapolis, MN, USA); IDE (rabbit IgG, 1:5,000, Abcam); and β-actin (mouse IgG, 1:400, Santa Cruz) . The following secondary antibodies were used: goat anti-mouse IgG/HRP (1:5,000, Golden Bridge International, Beijing, China); goat anti-rabbit IgG/HRP (1:5,000, Golden Bridge International); rabbit anti-goat IgG/HRP (1:5,000, Golden Bridge International). The intensity of the bands was quantified by using Image J software developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA.
Aβ40 and Aβ42 enzyme-linked immunosorbent assays (ELISAs) were performed by using ELISA kits (Invitrogen, Carlsbad, CA, USA). Aβ standards (1 to 40 or 1 to 42 aa) were used according to the manufacturer’s manual. Hippocampus and cortex from one hemisphere were homogenized in eight volumes of ice-cold guanidine buffer (5.0 M guanidine-HCl 50 mM Tris–HCl, pH 8.0) (six mice per group). The homogenate was incubated at room temperature for 4 hours and diluted at 1:20 with ice-cold reaction buffer BSAT-DPBS (Dulbecco phosphate-buffered saline, with 5% BSA, 0.03% Tween-20, 0.2 g/L KCl, 0.2 g/L KH2PO4, 8.0 g/L NaCl, 1.150 g/L Na2HPO4, pH7.4) containing 1 × Protease Inhibitor Cocktail (PMSF, aprotinin, leupeptin, EDTA, pepstatin A, NaF, and NaVO3), and then was centrifuged at 15,000 g at 4°C for 20 minutes. The supernatant was used for ELISA assay.
Quantitative real-time PCR
Total RNA from cortex and hippocampus of one hemisphere was extracted by using Trizol (Invitrogen) according to the manufacturer’s protocol (six mice per group); 1 μg total RNA was used for reverse transcription in a final volume of 20 μl. Reverse transcription was performed according to the manufacturer’s protocol (DRR047A, Takara, Otsu, Shiga, Japan). Then 2 μl cDNA was used for real-time PCR with the SYBR Premix Ex Taq (DRR041A, Takara, Japan). Quantitative real-time PCR was performed under the following conditions: 95°C for 30 seconds, 95°C for 5 seconds, 60°C for 34 seconds, and 40 cycles. All PCR reactions were performed in triplicate. The fold changes of target genes were calculated by using the delta delta cycle threshold (two delta delta CT) method . GAPDH was used as the reference gene. The PCR primers used in the study were reported previously .
All data were presented as mean ± standard error of the mean (SEM). Differences were analyzed with ANOVA. A value of P < 0.05 was considered statistically significant. In two-variable experiments, two-way ANOVA followed by Bonferroni post hoc tests were used to analyze the significance of differences between groups. One-way ANOVA was used to analyze one-variable experiments with three groups. The Student t test was used to compare two groups. Data were analyzed with the software SPSS 16.0 (SPSS Inc., Chicago, IL, USA).