Cell origin
Adipose tissue was collected by veterinarians at veterinary clinics. All dogs were privately owned patients and were under general anesthesia for other procedures, not purposefully to collect samples for this study. All dogs’ owners gave their written consent to use the removed adipose tissue for research purposes and all the experiments were performed according to the procedures and guidelines approved by the National Health Service branch of the Slovenian Ministry of Health. Additional ethical permission was not needed according to Slovenian legislation and interpretation by Administration for safe food, veterinary and plant protection, which is responsible for issuing licenses for experiments with animals. Subcutaneous adipose tissue (<1 cm3) was aseptically removed from the back area between the dog's scapulae. Samples were obtained from six different dogs aged 2 to 9 years (mean age 5.5 years) of different breeds and both sexes, three males and three females.
Isolation of canine ASCs
Canine ASCs were isolated from adipose tissue and characterized based on the routine protocols developed by the Animacel Ltd. Briefly, fat tissue was minced, washed three times in PBS buffer and digested over night with an equivalent amount of collagenase I solution containing 2 mg/ml collagenase I and 4 mg/ml BSA in HEPES buffer, pH 7.4. Collagenase I activity was stopped by a double volume of PBS and the resulting cell suspension was filtered through the falcon strainer (pore size 100 µm). Cells were grown in the cultivation medium, which was previously demonstrated to be specific for canine ASCs cultivation (32) and was developed by Animacel Ltd. After 24 hours non-adherent cells were washed off. ASCs were cultured until 80% confluence was reached, detached, counted with Bürker Turk Chamber hemocytometer and passaged. Again, cells were maintained until 80% confluence, detached and cryopreserved in a freezing medium consisting of DMEM Glutamax, FBS and DMSO, for later use.
Cultivation of ASCs
The high cell density is a key parameter for the successful differentiation of multipotent mesenchymal stromal cells into neural lineages. Isolated ASCs (first passage) were plated into different growth flasks depending on the designed experiment. During the optimization and monitoring of the morphology of undifferentiated and differentiated cells, two T25 flasks were used for cells from each dog, plated at a density of 106 cells/cm2. Three additional T25 flasks were required for flow cytometry, for each point of differentiation and for each animal, with the cells at the same density. Furthermore, for the immunocytochemical characterization of differentiated ASCs, individual round dishes with 35 mm diameter were used with cells at a density of 8x104 cells/cm2 and 24-well plates with cells plated on glass cover slips coated with laminin at a density of 2x104 cells/cm2.
Neural differentiation
Canine ASCs in a second passage were plated in different flasks based on the type of the experiment. Cells were grown in our standard cultivation medium. After 24 hours, the cultivation medium was replaced by the pre-differentiation neural induction medium (mitogenically stimulated; STIM). Two pre-diffrentiation media were tested. STIM1 consisting of DMEM Glutamax, EGF (20 ng/ml), bFGF (20 ng/ml) and 1x B27 supplement (supplied as 50x stock solution, Gibco); and STIM2 consisting of DMEM Glutamax, β-mercaptoethanol (100 µM) and 20% FBS. After 24 hours, the pre-differentiation medium was replaced by the neural differentiation medium. Four different differentiation media were prepared:
- KEM media consisting of DMEM Glutamax, 1% FBS, 2x B27 supplement, valproic acid (2mM), forskolin (10 µM), hydrocortisone (1 µM), KCl (5 mM), butylated hydroxyanisole (BHA; 200 µM), insulin (5 µg/ml) and 0.1% penicillin/streptomycin.
- NIMa (neural induction medium a) composed of DMEM Glutamax, 2x B27 supplement, 1x N2-supplement, 1% FBS, 0.1% penicillin/streptomycin and 10 nM retinoic acid.
- NIMb consisting of DMEM Glutamax, 2x B27 supplement, 1x N2-supplement, 1% FBS, 0.1% penicillin/streptomycin and 100 nM retinoic acid.
- NIMc composed of DMEM Glutamax, 2x B27 supplement, 1x N2-supplement, 1% FBS, 0.1% penicillin/streptomycin and 10 µM retinoic acid.
The cells were further processed for morphological, immunocytochemical and flow cytometry analysis after 3, 6 and 9 days following neural induction.
Viability assay
To determine the cell numbers and the viability of canine ASCs cells after exposure to neural differential medium, dye exclusion assay was used. The viability was determined at three time points (after 24 hours of pre-differentiation in STIM1 medium and at 3 and 9 days after induction of differentiation) by adding Trypan Blue dye into cell suspension and counting live/dead cells using a Bürker Turk Chamber under the light microscope. The viability of cells grown in control media was determined for comparison to neurally differentiated cells. All experiments were performed in triplicates.
Immunohistochemistry on dog brain
Dog brain tissue sections were used as control for the reactivity of antibodies. Dog brain was obtained at Veterinary faculty of University of Ljubljana following the dog’s owner’s approval. The dog was sixteen years old male, euthanized due to advanced cognitive dysfunction. The brain was surgically removed, cut in small cubes and fixed in 4% paraformaldehyde at 4 °C for several days. Pieces of the brain were embedded in paraffin using an automated tissue processor (Tissue processor Leica TP 1020). Tissue blocks containing the area of the frontal cortex were cut in 7 µm sections and further processed for immunohistochemistry. After dewaxing sections were subjected to antigen retrieval in sodium citrate buffer (10 mM sodium citrate, 0,05% Tween 20, pH 6) by boiling the slides for 10 minutes in a microwave oven. This was followed by blocking the unspecific epitopes in 1.5% BSA, 10% normal goat serum, 0.1% TritonX-100 in PBS for 60 minutes at room temperature. Sections were then incubated overnight at 4 °C with the following primary antibodies: mouse anti-GFAP (1:400, G3893, Sigma), mouse anti-NF-H (1:400, AB1989, Millipore), mouse anti-bIII tubulin (1:400, sc-80005, Santa Cruz Biotechnology), rabbit anti-MAP2 (1:400, AB5622, Millipore) and chicken anti-nestin (1:200, ABIN187958, Neuromics Antibodies). The next day slides were washed with PBS for 5 min and incubated with secondary antibodies (anti-rabbit Alexa Flour 555 or anti-mouse Alexa Flour 555 diluted 1:1000 in blocking buffer (both from Invitrogen)) for 1h at room temperature in the dark. The nuclei were counterstained with DAPI (Sigma). Slides were then washed with PBS thrice for 5 minutes and mounted in ProLong Gold Antifade Mountant (Molecular Probes). Stainings were visualized with a confocal microscope (Zeiss LSM 710) and ZEN software.
Immunofluorescence analysis of cells
Immunocytochemistry was performed on cells to evaluate the presence of the neural markers after the induction with differentiation medium. One 24-well plate was prepared per each time point with round glass cover slips coated with laminin (10 µg/ml). Next day, the second passage canine ASCs were plated at a density of 2x104 cells/cm2 in 1 ml of the basal growth medium. At 3 and 9 days of differentiation cells were fixed with 4% paraformaldehyde (pH 7.4) for 10 min at room temperature, rinsed three times in PBS and permeabilised with TBST for 5 min. Blocking of unspecific epitopes was performed with blocking solution (10% FBS, 1% milk powder, 0.02% Na-azide, TBST, pH 7,2) for 1h. Cells were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-GFAP (1:400, G3893, Sigma) conjugated with Alexa Flour 488, rabbit-anti-NF-H (1:400, AB1989, Millipore) and rabbit anti-bIII tubulin (1:1000, AB9354, Millipore). The next day cells were rinsed three times with PBS and one time with 2% FBS in PBS and incubated with secondary donkey anti-rabbit Cy2 conjugated IgG antibody or secondary donkey anti-mouse Cy3 conjugated IgG antibody (both 1:500; Jackson Immunoresearch) for 2h at room temperature in dark. Anti-GFAP antibody was covalently labelled with mouse anti-rabbit Alexa Flour 488 IgG using Alexa Fluor® 488 Protein Labeling Kit (Molecular Probes). The nuclei were counterstained with DAPI. Stainings were visualized with a confocal microscope (Zeiss LSM 710). Neural differentiation was detected by observing random viewing fields under the microscope and by comparing cellular morphology. NF-H and TUBB3 positive cells were identified as neuronal lineage and GFAP-positive cells were identified as glial lineage (astrocytes).
Flow cytometry
ASCs cell cultures were detached for flow cytometry analyses at three time points (pre-differentiation, 3 and 9 days of differentiation). Confluent cells were harvested using trypsin (TrypLE™ Express, Gibco) digestion for 5 min at 37 °C, centrifuged for 5 min at 1400 rpm at 4 °C and resuspended at a concentration of 107 cells/ml in PBS. Five aliquots were prepared per time point (pre-differentiation, 3 and 9 days of differentiation) per animal each containing 100 µl (concentration of 106 cells/ml) and were transferred to flow cytometry facility at Medical Faculty, University of Ljubljana. The aliquots were centrifuged for 5 min at 1600 rpm at room temperature and pellet was fixed with 4% paraformaldehyde (pH 7.4) for 10 min at room temperature, rinsed three times in PBS, and in parallel permeabilized with 0.1% Triton X-100, and blocked with 2% FBS, 5% milk powder, 0.02% penicillin/streptomycin for 10 min. Cells were incubated for 30 min in the dark at 37 °C with following primary antibodies: rabbit anti-NF-H (1:400, AB1989, Millipore), rabbit anti-MAP2 (1:200, AB5622, Millipore) and mouse anti-GFAP (1:400, G3893, Sigma) conjugated with Alexa Flour 488 (Life Technologies). After incubation samples were centrifuged 5 min at 1600 rpm, rinsed two times with 2% FBS in PBS and the ones stained for NF-H and MAP2 incubated with secondary donkey anti-rabbit IgG Cy2 conjugated antibody (1:500, Jackson Immunoresearch). Anti-GFAP antibody was previously labelled as described above. All samples were analyzed by BD FACS Canto flow cytometer (BD Biosciences). Appropriate isotype matched controls conjugated to FITC were used to identify nonspecific staining for GFAP antibody. For unconjugated primary antibodies, controls included isotype matched unconjugated primary controls and incubation with the secondary antibody alone. Data was analyzed using FACSDiva™ version 6.1.2 (BD Biosciences) analysis software.
Statistical analysis
Immunocytochemical staining and cell morphology were evaluated only qualitatively by observing random viewing fields by observer blind to the differentiation conditions. Differences in cell numbers and cell viability between control and differentiated ASCs were analyzed by repeated measures ANOVA followed by Bonferroni post-hoc test with sex as independent variable and day of culture as within factor. Differences between groups in expression of neuronal markers detected by flow cytometry were analyzed by ANOVA followed by Bonferroni post-hoc test. All differences were considered statistically significant at P < 0.05.