Primary neurosphere culture
Two-month-old C57/Bl6 wild-type mice were euthanized and their brains were removed and placed into sterile Dulbecco's modified Eagle's medium/F12 (DMEM/F12). A coronal slice (approximately 1 mm) was dissected starting 1 to 2 mm posterior to the olfactory bulb. The region occupying the lateral wall and anterior horn of the lateral ventricles was removed with the aid of a dissecting microscope and diced with a sterile scalpel. Neurosphere culture was prepared as previously described . Briefly, tissue pieces were collected in a mixture of Papain and DNase in Earl's balanced salt solution and incubated at 37°C for 40 minutes. Then, tissue pieces were pelleted by centrifugation and dissociated to a single-cell suspension, and cells were plated in complete medium-water, DMEM/F12 (Gibco, now part of Invitrogen Corporation, Carlsbad, CA, USA), glucose (Sigma-Aldrich, St. Louis, MO, USA), NaHCO3 (Sigma-Aldrich), HEPES (Sigma-Aldrich), L-glutamine (Invitrogen Corporation), penicillin/streptomycin (Invitrogen Corporation), putrescine (9.6 μg/mL; Sigma-Aldrich), apotransferrin (0.1 mg/mL; Sigma-Aldrich), insulin (0.025 mg/mL; Roche, Indianapolis, IN, USA), selenium (5.2 ng/mL; Sigma-Aldrich), progesterone (6.3 ng/mL; Sigma-Aldrich), bovine serum albumin (BSA) (2 mg/mL; Sigma-Aldrich), heparin (4 μg/mL; Sigma-Aldrich), EGF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA), and bFGF (10 ng/mL; PeproTech)-and passaged after 10 days.
Isolation of mesenchymal stem cells
After euthanasia, the bone marrow contents of the femurs and tibia of donor Balb/C mice were flushed through a 40-μm filter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) into a 50-mL tube (Corning, Corning, NY, USA) containing MSC media: 40% alpha-modified Eagle's medium (Invitrogen Corporation, Rockville, MD, USA), 40% F-12 nutrient mixture (Invitrogen Corporation), 10% fetal bovine serum (Valley Biomedical, Winchester, VA, USA), and 1% antibiotic-antimycotic solution (Invitrogen Corporation). Bone marrow cells were plated at a density of 20 × 106 per 9.6 cm2 in MSC media at 37°C in 5% CO2 as previously described . The non-adherent population was removed after 72 hours, and the adherent cells were washed with fresh media and cultured for 7 days. The resulting adherent cells were harvested by incubating with 0.25% trypsin (Invitrogen Corporation) followed by gentle scraping. By means of negative selection via immunomagnetic column (Miltenyi Biotec, Auburn, CA, USA), cells negative for CD11b (eBioscience, San Diego, CA, USA) and CD45 (eBioscience) were placed back into culture. A homogenous cell phenotype was confirmed on the basis of the expression of CD29, CD44, and Sca1 and the absence of hematopoietic (CD45, CD14, and CD11b) markers. Prior to use, cells had been passaged from one to four times.
Human decidua parietalis placenta stem cells
All studies were approved by the Institutional Review Board of the University of Illinois. hdpPSCs were isolated from the decidua parietalis dissected from placental membranes after normal vaginal delivery at term, as previously described in detail . Human placenta tissue was obtained from the Human Female Reproductive Tissue bank in the Center for Women's Health and Reproduction at the University of Illinois at Chicago. Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated and charcoal-stripped fetal bovine serum, 0.1 mM sodium pyruvate, and 1% penicillin/streptomycin. At confluence, cells were trypsinized, propagated, and used for experiments in passage numbers three to five.
sAPPα (Sigma-Aldrich) was used at 10 nM concentrations unless otherwise indicated (dissolved in phosphate-buffered saline, or PBS).
Neurosphere media was conditioned by plating 3 × 105 NPCs in each well of a 12-well plate in 500 μL of complete media. After 1 hour, media was removed and spun at 1,000g for 10 minutes to remove any cells or debris. For depletion of sAPP, conditioned media was precleared with protein A-agarose beads (Pierce, Rockford, IL, USA) and then incubated overnight at 4°C with 22C11 antibodies against the N-terminus of APP or IgG antibodies (Millipore Corporation, Billerica, MA, USA) as a control. Protein A-agarose beads were added for 30 minutes, the mixture was spun at 4,000 revolutions per minute (rpm) for 3 minutes, and the supernatant was used as depleted media. Regular conditioned media was subjected to the same process without antibody incubation as a control. All media was filtered through a 0.22-mm filter prior to addition.
Detection of sAPP
For the detection of soluble APP from brain lysates, protein was extracted in immunoprecipitation buffer containing 150 mM NaCl, 50 mM Tris-Cl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton-X 100, 0.5% sodium deoxycholate, protease inhibitor cocktail, and 250 μM phenylmethylsulfonyl fluoride (PMSF). To remove full-length APP, protein samples were immunodepleted by using 369 antibodies against the C-terminus of APP (a gift from Sangram S Sisodia, The University of Chicago). Briefly, samples were precleared with 50 μL of immobilized protein A-agarose beads (Pierce) at 4°C for 30 minutes. Samples were centrifuged at 4,000 rpm for 3 minutes, and the pellet was discarded. To the supernatant, 5 μL of 369 antibody was added and incubated overnight at 4°C. The next morning, 50 μL of immobilized protein A was again added for 30 minutes at 4°C and spun at 4,000 rpm for 3 minutes. The pellet contains the full-length APP-369 antibody complex, and the supernatant was probed for sAPP by using 22C11 antibodies raised against the N-terminus of APP (Millipore Corporation).
Neurosphere formation (clonogenic) assay
Briefly, neurospheres were singly dissociated by mechanical dissociation and plated at 1,000 cells per well onto 96-well plates. For matrix-metalloproteinase (MMP) inhibitor experiments, cells were then treated with the indicated molar concentration of GM6001 or GM6001 negative control (Millipore Corporation) and the indicated molar concentrations of sAPP or conditioned media. If not otherwise indicated, 1 μM GM6001 and negative control inactive inhibitor (NC) were used. Cells were treated every 72 hours for 10 days. After 10 days in culture, neurospheres were counted under an inverted light microscope, and the average neurosphere diameter was calculated from 25 randomly assigned squares of the grid by using a Zeiss AX10 microscope (Carl Zeiss Ltd., Hertfordshire, UK) and StereoInvestigator software (MBF Bioscience, Williston, VT, USA). After sphere size determination, cells were singly dissociated with a p200 pipette and counted with a hemocytometer. The remaining cells were placed onto Matrigel-coated chamber slides for 30 minutes and then fixed in 4% paraformaldehyde for 30 minutes for immunocytochemistry. Briefly, cells were washed four times in Tris-buffered saline (TBS) and then placed into blocking solution (5% normal donkey serum, 0.25% Triton-X 100 in TBS) for 30 minutes at room temperature. Next, cells were incubated in primary antibodies-mouse anti-nestin (1:100; Millipore Corporation) and goat anti-Sox2 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)-in TBS containing 0.25% Triton-X 100 for 1 hour at room temperature. After primary antibodies, cells were again incubated in blocking solution for 30 minutes at room temperature before secondary antibody incubation-anti-goat cy5 (1:250; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and anti-mouse cy3 (1:500; Jackson ImmunoResearch Laboratories, Inc.)-in TBS with 0.25% Triton-X 100 for 30 minutes at room temperature in the dark. Cells were then washed four times in TBS and incubated for 5 minutes with DAPI (4'-6-diamidino-2-phenylindole) (1:50,000; Invitrogen Corporation) at room temperature in the dark. Cells were then washed three times and mounted with polyvinyl alcohol-DABCO mounting solution. Cell counts were made by means of StereoInvestigator software version 8 (MBF Bioscience).
Mesenchymal and placental cell proliferation experiments
MSC or hdPSC were trypsinized for 5 to 10 minutes with 0.05% trypsin, collected after trypsin inactivation, and spun at 500g for 5 minutes. Pellets were then dissociated and plated at 1,000 cells per well in 96-well plates. Cells were then treated with the indicated molar concentration of GM6001 or GM6001 negative control (Millipore Corporation) and the indicated molar concentrations of sAPP. After 3 days in culture, cells were trypsinized for 5 to 10 minutes, spun, and counted with a hemocytometer.
Brain tissue for biochemistry and immunohistochemistry
Experiments using animals were performed according to guidelines of the National Institutes of Health and the University of Illinois at Chicago Institutional Animal Care and Use Committee. The APP knockout [APP(-/-)] model has been described previously [23, 24]. Briefly, the authors generated the mice through homologous recombination in embryonic stem cells. Mice heterozygous for APP expression were cross-mated and APP(+/+) (APP wild-type), APP(+/-), and APP(-/-) resulted from this breeding. Our colony is maintained via group housing (fewer than five mice per cage) in a barrier facility under a 14:10 light/dark cycle with free access to food and water. Animal care and procedures were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Brain tissue processing
For in vivo immunohistochemical staining, male APP(+/+) mice from 3 to 5 months old were used. All mice were anesthetized with a mixture of ketamine and xylazine and transcardially perfused with 100 mL of ice-cold PBS. The brains were then removed and halved in the sagittal plane. The left half was immediately placed into 4% paraformaldehyde on ice. From the right half of the brain, the following regions were dissected for biochemical analysis and immediately placed into Eppendorf tubes on dry ice: SVZ, hippocampus, olfactory bulb, frontal cortex, and cerebellum.
Left hemibrains from PBS-perfused mice were post-fixed in 4% paraformaldehyde for 3 days and stored in 30% sucrose at 4°C. Hemibrains were sectioned sagittally at 50 μm by using a microtome and placed into cryopreservent (47.6% PBS, 28.57% ethylene glycol, and 25% glycerin vol/vol). Sections were blocked by using a solution containing 0.25% vol/vol Triton-X 100 (Sigma-Aldrich) and 5% vol/vol Normal Donkey Serum (Jackson ImmunoResearch Laboratories, Inc.) in TBS. The following antibodies were used: Dlx-2 (1:200; Millipore Corporation), nestin (1:100; Millipore Corporation), Sox2 (1:100; Santa Cruz Biotechnology, Inc.), ADAM10 (1:200; Millipore Corporation), and APP (22C11; Millipore Corporation and A8717; Sigma-Aldrich). Floating sections were incubated in primary antibodies for 72 hours at 4°C before continuing with blocking, biotin conjugation (Jackson ImmunoResearch Laboratories, Inc.), and secondary antibody incubation (cy2 Streptavidin, anti-mouse cy3, anti-goat cy5, and anti-rabbit cy5; Jackson ImmunoResearch Laboratories, Inc.).
Protein extraction from brain tissue was performed in lysis buffer containing 1X TNE, 50 mM Tris, 150 mM NaCl, 5 mM EDTA, protease inhibitor cocktail (Sigma-Aldrich), and 100 mM PMSF. Quantification of protein was performed by using the bicinchoninic acid (BCA) method (Pierce), and equal amounts of protein were subjected to direct immunoblotting. For the extraction of protein from neurosphere, mesenchymal, and Human decidua parietalis placenta stem cells cultures, a lysis buffer containing 150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, 1% Triton-X 100, 0.5% sodium deoxycholate, protease inhibitor cocktail, and 250 μM PMSF was used. For quantification, at least three cultures were used.
Erk and Akt signaling
To assay phosphorylation of Erk and Akt, neurospheres were singly dissociated and plated at 5 × 105 cells per well in a six-well plate in Earle's balanced salt solution (Sigma-Aldrich) and treated immediately with 1 μM GM6001 or GM6001 negative control. After 1-hour incubation at 37°C, one of the GM6001-treated groups was subsequently treated with 10 nM recombinant sAPPα (Sigma-Aldrich) for 15 minutes before all groups were lysed in ROLB buffer: 10 mM HEPES, pH 7.4, 0.5% Triton X-100, 80 mM β-glycerophosphate, 50 mM sodium fluoride, 2 mM sodium orthovanadate, 100 nM staurosporine, 100 nM K252a, 50 nM okadaic acid, 50 nM microcystin, mammalian protease inhibitor cocktail (Sigma-Aldrich), and phosphatase inhibitor cocktail II (Calbiochem, now part of EMD Biosciences, Inc., San Diego, CA, USA) in water. After lysis, protein quantification was performed by using the BCA method (Pierce), and equal amounts of protein were run on Tris-glycine gels and transferred to nitrocellulose membranes. For blocking and antibodies, we employed a solution of 0.05% vol/vol Tween, 10% wt/vol milk, and 0.1% wt/vol BSA (Sigma-Aldrich) in TBS. The following antibodies were used: pErk (1:500; Santa Cruz Biotechnology, Inc.), Erk (1:1,000; Santa Cruz Biotechnology, Inc.), pAkt (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), and Akt (1:500; Cell Signaling Technology, Inc.). (N = 3 for Erk and Akt Western blot quantification.)