hAT-MSCs: sources, culture and characterization.
The cells were obtained from commercial and human sources
1a. Commercial hAT-MSCs.
Cells were obtained from the POIETICS bank - Adipose-Derived Stem Cells (cat. #PT-5006, donor 34464. The cell type was confirmed by the presence of clusters of differentiation (CDs) such as CD13, CD29, CD44, CD73, CD90, CD105 and CD166, and by the absence of CDs such as CD14, CD31 and CD45. They were tested negatively for mycoplasma, bacteria, yeast and fungi by the supplier company. A frozen vial containing ~ 1x106 cells was thawed at 37ºC and plated in 25 cm2 flask (TPP). Cells were cultured with DMEM medium (Sigma) containing 10% FBS (Cripion), 100 units/mL penicillin (Gibco), 100 µg/mL streptomycin (Gibco), 50 mg/L gentamicin (Sigma) and 2.5 mg/L fungizone (Sigma). After 24 hours, debris and non-adherent cells were gently removed [36]. When adherent cells reached 80% confluence (passage1: P1), hADSCs were detached with 0.25% trypsin/1 mM ethylenediamine tetra acetic acid (EDTA) (Sigma) and plated in flasks at a density of 1,5×104 cells/75 cm2 (passage 2: P2). The cellular density was determined by manually counting the number of cells at each passage[38]. The cells are named as cell 0 (C0). C0 were expanded under the above-described conditions and used only from 4th to 8th passage [29, 38, 39].
1b. Patient-derived hAT-MSCs
Cells were obtained from the subcutaneous adipose tissue of two 30-year-old females submitted to abdominal liposuction at the Hospital de Clínicas in Porto Alegre (University Hospital), RS, Brazil. The patients agreed to participate in the research and signed a consent form (GPPG 2018 − 0374). The fresh adipose tissue was washed with PBS buffer, minced and digested for 1 h in 0.1% collagenase at 37°C. The digestion process was stopped by the addition of Dulbecco's Modified Eagle's Medium (DMEM) containing 20% fetal bovine serum (FBS) and 100 units/mL penicillin (Gibco), 100 µg/mL streptomycin (Gibco). The digested suspension was filtered through a 70 µm nylon mesh cell filter to retain the tissue debris. The filtered suspension was centrifuged at 400×g for 5 min. The stromal vascular fraction (pellet) was resuspended in DMEM + 20% FBS (Cripion) medium and cultured in flasks cell culture of 25 mc2 (TPP) at 37°C in a humidified atmosphere of 5% CO2. After 24 hours, the non-adherent cells were gently removed[38]. When adherent cells reached 80% confluence (passage 0: P0), confluent cells (hAT-MSCs) were detached with 0.25% trypsin/1 mM ethylenediamine tetra acetic acid (EDTA) (Sigma) and plated in flasks at a density of 1,5×104 cells/75 cm2 (TPP) (passage1: P1). Cells were cultured with DMEM medium (Sigma) containing 10% FBS (Cripion) and 100 units/mL penicillin (Gibco), 100 µg/mL streptomycin (Gibco), gentamicin 50mg/L (Sigma) and fungizone 2.5mg/L (Sigma). These cells are named as cell 1 – patient 1 (C1) and cell 2 – patient 2 (C2). C1 and C2 were expanded under the same above-described conditions and used only from 4th to 8th passage[35, 39].
C1 and C2 hAT-MSCs were characterized by immune fluorescence through flow cytometry and confocal microscopy.
Flow cytometry: hAT-MSCs were centrifuged (400×g for 5 min at room temperature), the cell pellet was resuspended in DMEM + 10% FBS and the cells were counted in a Neubauer chamber. Shortly after, cells were incubated with antibodies at concentration of 1:50 for 4 h at 37°C. Then, cell suspensions were centrifuged at 400×g for 5 min at room temperature, and cell pellets resuspended in 200 µl of PBS. Ten thousand events were analyzed using flow cytometry (BD FACSCalibur™)[40]. Cells, only in passage 4 (P4), were characterized as hAT-MSCs by CDs presence: CD34 (FITC Mouse Anti-human CD34 BD Pharmingen), CD45 (Human CD45 FITC Conjugate, Invitrogen), CD90 (PE Mouse Anti-Human CD90 BD Pharmingen) and CD105 (Huan CD105 R-PE conjugate, Invitrogen).
Confocal microscopy: an aliquot of 1x104 hAT-MSCs was placed on a slide and analyzed by immunofluorescence. Cells were maintained in culture conditions for 72 h to adhere to the coverslip. Then, cells were incubated for 4 h at 37°C with the same antibodies used for cytometry: CD34, CD45, CD90 and CD105, at a ratio of 1:500. The negative control was prepared by incubating only the secondary antibodies: Alexa Fluor 555 (Invitrogen) and Alexa Fluor 488 (Invitrogen). Cells were gently washed in the coverslip with PBS (4 times) to remove excessive antibodies, followed by fixation with PFA 4% for 2 h. Cells were gently washed again with PBS, and the coverslips fixed with Fluoromount (Sigma) onto a histological slide for further analysis. Images were acquired using an 8-bit gray scale confocal laser scanning microscope (Olympus FV1000). Approximately 10−15 sections with 0.7 µm thick confocal were captured parallel to the coverslip (XY sections) using a ×20 objective (Olympus, U plan-super-apochromat, UPLSAPO 60X). Z-stack reconstruction and analysis were conducted using ImageJ software (http://rsb.info.nih.gov/ij/).
2. Extracellular Vesicles (EVs)
2a. EVs isolation and purification
As cultured hAT-MSCs (P4-P8) reached 80% confluence, DMEM + 10% FBS medium was replaced by DMEM FBS-free (to avoid isolated vesicles contamination by FBS proteins). After 72 h of culture, the medium was collected for vesicles isolation and cells remained in culture. To recover from stress caused by FBS removal, the remained cells were supplemented with DMEM + 10% FBS for 72 h[29].
For EVs isolation, the medium was collected and centrifuged (3 times) at 4°C: (1st : 400×g for 15 min, 2nd : 2000×g for 15 min and 3rd : 10,000×g for 30 min). The supernatants were filtered through a 0.22 µm membrane. The isolation was finished by centrifugation (100,000×g at 4°C for 2 h). The supernatant was discarded, PBS was used to wash the pellet containing EVs, and the cell suspension was centrifuged at 100,000×g at 4°C for 2 h[41]. Finally, the pellet was resuspended in 100 µl of PBS and stored at − 20°C[38]. EVs protein content was quantified by bicinchoninic acid (BCA) assay (Thermo Scientific Pierce™)[38]. The vesicles isolated from C0, C1 and C2 cells are named EV0, EV1 and EV2, respectively.
2b. EVs Characterization
EVs were characterized by flow cytometry and identification of membrane proteins CD63 and CD81 presence[42]. Firstly, EVs were incubated with magnetic beads (Thermo Fisher - Scientific - Invitrogen™) coated with primary antibody CD63 (Exosome-Human CD63, Thermo Fisher - Scientific - Invitrogen™) and CD81 (Exosome-Human CD81, Thermo Fisher - Scientific - Invitrogen™) for 18 h at 4°C under gentle stirring. For each preparation, 10 µl of 1 mg/ml EVs suspension was applied. To remove excess of beads, EVs were washed with PBS: 2mL of PBS was added for 5 minutes, then the tub was placed in a magnet for 1 minute and the supernatant was discarded. Then, CD63 (CD63 Anti-human Mouse, FITC, Clone: MEM-259, Invitrogen ™) and CD81 (PE Anti-Human Mouse CD81 Clone JS-81, BD Pharmingen ™) antibodies (without granules) were added to the solution containing the EVs + magnetic beads. After 1 h of incubation, the EVs were gently washed by placing the tube on a magnet for 1 minute, discarding the supernatant. We added 2mL of PBS (to remove excess antibody) for 5 minutes and again placed the tube on a magnet for 1 minute and discarded the supernatant. Finally, the EVs were resuspended in 200 µl of PBS for analysis. Ten thousand events were analyzed by flow cytometry.
To measure the particle size and the polydispersity index (PDI) we used photon correlation spectroscopy. The EVs suspension derived from hAT-MSCs (50 µl) at 1 mg/ml was diluted in 1 ml of PBS. All analyzes were performed in triplicate using a Malvern Nano-ZS90® (Malvern Instruments, England) at 25 ° C.
2c. EVs Purity Measurement
Transmission electron microscopy (TEM) analysis, using direct examination technique, was used to evaluate EV purity and diameter sizes[37]. EVs suspension (10 µL), 1 mg/ml of protein, was aliquoted onto a grid covered with carbon film (formvar/carbon) and dried at room temperature. Uranyl was used as a contrast. The sample was analyzed by TEM 120Kv (JEM 1200 Exll-JEOL).
2d. EVs Labeling
EVs were labeled with red fluorescent membrane dye PKH26 (MINI26, Sigma). In brief, the EVs-containing PBS solution was centrifuged at 100,000×g for 2 h, at 4°C and the pellet suspended with the diluent of the fluorescent kit. Filtered PKH26 (4mM) and EVs (200 µg/ml) were mixed at a ratio 1:1 for 5 min, followed by the addition of 5% BSA. To remove excess dye, the EVs were washed (3 times): we added 5 ml of PBS and centrifuged at 100,000×g for 2 h, at 4°C, discarded the supernatant. In the last centrifugation, stained EVs pellet was suspended in 0.5 mL of PBS. To eliminate any dye aggregates, the solution was filtered through a 0.2 µm membrane filter [35].
3. Animals
3a. Characterization
Adults (90–120 days old) male Wistar rats weighting 350–400 g were maintained under controlled light (12/12 h light/dark cycle), 22°C ± 2, with water and food ad libitum.
All procedures were performed according to the Guide for the Care and Use of Laboratory Animals and to the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal studies.
The Ethics Committee for the Use of Animals at the Universidade Federal do Rio Grande do Sul (process number: 31888) approved this project.
The schematic procedures are illustrated in Fig.
1.
3b. Focal Permanent Ischemia and Sham Procedures
Anesthetized animals (ketamine hydrochloride: 90 mg/kg, 450 µl/kg i.p. and xylazine hydrochloride:10 mg/kg, 300 µl/kg i.p.) were placed into a stereotaxic apparatus. After skin incision, the skull was exposed, and the craniotomy was performed by exposing the left frontoparietal cortex (+ 2mm to − 6mm A.P. and − 2mm to − 4mm M.L. from the bregma). A focal permanent ischemic lesion was induced by thermo-coagulation of motor and sensorimotor pial vessels [43–47]. Blood vessels were thermo-coagulated by placing a hot probe near the dura mater for 2 min, until red-brown color indicated complete thermo-coagulation. Soon after, the skin was sutured, and animals placed on a heating pad at 37°C, until full recovery from anesthesia. Animals from sham group were only submitted to the above-mentioned craniotomy. Animals were randomly allocated to 3 treatment groups: Sham; Ischemic (ISC); Ischemic treated with EVs (ISC + EV).
3c. Intranasal EVs Treatment
Intranasal EVs treatment was performed 24 h after ischemic or sham procedure. Sedated animals (O2 flow rate at 0.8-1.0 L/min with Isoflurane levels of 2.5-3.0 %) slowly (during 20 sec) received, into the nasal cavity, a single 50 µL of EVs (ISC + EVs) or 50 µL PBS (Naive, Sham, ISC). The 200 µg/kg EVs dose was selected based on a dose/effect curve: ISC + PBS; ISC + 100 µg/kg; ISC + 200 µg/kg and ISC + 300 µg/kg (n = 3).
4. Brain analysis
4a. Extracellular Fluorescent Vesicles (EVs) Detection in Rat Brain
Distribution of EVs in rat brains was analyzed 18 h after intranasal administration of fluorescent EVs (PKH26-mini, Sigma)[32, 35, 37]. Anesthetized animals (ketamine hydrochloride: 90 mg/kg, 450 µl/kg i.p. and xylazine hydrochloride: 10 mg/kg, 300 µl/kg i.p.) were transcardially perfused using a peristaltic pump with PBS followed by perfusion with PFA 4% (both 10 mL/min, 100mL). Brains were dissected and immersed in PFA 4%, pH 7.4 and stored for a maximum of 7 days at 4°C. Coronal brain Sect. 20 µm thick were obtained using a vibratome (Leica) at + 2.20 mm, 0,20 mm and − 1,88 mm of Bregma. Brain slices were mounted on glass slices and incubated for 5 min in the dark with 1 µg/mL Hoechst dye (33342 Sigma-Aldrich) in PBS to detect cell nuclei. The slices were washed with PBS (4 times) and the slices fixed with fluoro mount (Sigma).
Slices images for counting EVs were acquired using an 8-bit gray scale confocal laser scanning microscope (Olympus FV1000). Approximately 10−15 sections with 0.7 µm thick confocal were captured parallel to the coverslip (XY sections) using a ×60 objective (Olympus, U plan-super-apochromat, UPLSAPO 60X). Z-stack reconstruction and analysis to count the vesicles in the brain tissue were conducted using ImageJ software, briefly: background noise was removed using the “subtract background” tool. Images were converted to binary masks using default threshold option and vesicles were counted with the “analyze particles” tool (size = 0.05–0.90 µm). These settings were programmed into a macro and used for all analyzed images (http://rsb.info.nih.gov/ij/) (n = 3 naive and n = 5 ischemic for each group, 3 sections in each rat per group).
4b. Short term infarct volume
For the short-term evaluation of infarct volume, Naive, Sham, ISC, ISC + EV0 and ISC + EV2 groups were sedated 48 hours after treatment (O2 flow rate of 0.8 -1.0 mL/min with Isoflurane levels of 2.5-3.0%) and culled. Coronal sections of the whole brains were sliced at 2 mm, immersed in 2% 2,3,5-Triphenyl-tetrazolium chloride (TTC). After 30 minutes of incubation at 37 ºC, the slices were dipped in 4% buffered paraformaldehyde (pH 7.4) for 24 hours. The size of the infarct area was evaluated as the area devoid red staining. Infarct volume was measured using the imageJ software[44] (3 rats/group, 6 sections/rat).
4c. Brain Angiogenesis
After 42 days of treatment, animals from groups Naive, ISC and ISC + EV2 were anesthetized and received intracardial injection of 50 mg/mL (500 µl) fluorescein isothiocyanate-dextran amine (Merck) to label brain blood vessels. Rat brains were removed, immediately fixed in PFA 4%, and cut at 30 µm coronal slices in a vibratome. Images were acquired in fluorescence microscope (Nikon). The images were taken from the ipsilateral and contralateral sides in the Secondary Motor Cortex (M2) and somatosensory regions (SS) using the coordinates: +2.20 mm, 0.2 mm and − 1.88 mm A.P. to Bregma (PAXINUS online Rat Brain Atlas: http://labs.gaidi.ca/rat-brain-atlas/) (n = 3 per group). Blood vessels parameters as the total length (sum of length of segments, isolated elements and branches in the analyzed area) and the number of branches (in the analyzed area) were quantified with the Angiogenesis Analyser Plugin (Gilles Carpentier Research) ImageJ software (https://imagej.nih.gov/ij/).
5. BBB permeability
5a. Evans Blue in brain parenchyma.
Naive (n = 3), Sham (n = 3), ISC (n = 3) and ISC + EV2 (n = 3) animals were anesthetized 48 hours after treatment (ketamine hydrochloride – 90 mg/kg, 450 µl/kg i.p. and xylazine hydrochloride 10 mg/kg, 300 µl/kg i.p.) and received 3 ml/kg of Evans Blue (EB) solution 2% in saline, through the gingival artery (Supplementary information 1). After 1 hour, the animals were submitted to cardiac perfusion using a peristaltic pump (10 mL/min, with PBS, 100mL). Animals were culled, their brains were removed, weighed and whole brain was sliced at 2 mm for image acquisition for each slice. After, all slices together from each brain were macerated and homogenized in 2.5 ml of PBS and vortexed for 2 minutes. For the precipitation of proteins, 2.5 ml of 50% trichloroacetic acid was added to the homogenate, incubated for 12 hours at 50 ºC, centrifuged at 14,000 x g for 10 minutes. The concentration of the blue color was measured by a spectrophotometer (620 nm). EB dye was expressed in µg/g brain tissue against a standard curve [48, 49].
5b. CSF albumin levels
Albumin assay was performed using High Performance Liquid Chromatography coupled to Fluorescence detector (HPLC-FLD). The CSF method was validated according with the FDA guidelines [50, 51]. HPLC-FLD consisting of LC Shimadzu system (Kyoto, Japan) equipped with LC- 20AT pump, DGU-14A degasser, thermostat for CTO-10A column and Fluorescence detector, RF 20A was used. Acquisition and processing of the data was obtained using the LC Solution software. The FLD was set at 278 nm (excitation) and 335 nm (emission). Agilent reversed-phase ZORBAX SB-C18 column (5 µm particle size, 250 × 4.6 mm i.d.) was used. The method was performed using gradient condition, consisting of solvent A (H2O + 0.1% formic acid) and solvent B (acetonitrile (ACN) as follows: A → 65% B → 35% (0–5 min), A → 70% B → 30% (5–10 min), A → 65% B → 35% (10–17 min). The flow rate was set at 0.7 mL/min. Samples preparation was performed by adding to a 10 µL liquor in 40 µL of ACN and mixed in a vortex. The solution was transferred to conical vials and 10 µL was injected. Albumin stock solutions 1 mg/mL in water were stored at − 20 ± 2°C. For each day of analysis, standard solutions of albumin were prepared at 0,1, 0,5, 1, 10, 50 and 100 µg/mL.
6. Behavioral evaluation
6a. Cylinder Task (CT)
The cylinder task, which allows the evaluation of motor sequelae caused by ischemic insult[52], were used to determine animal motor symmetry of front paws. Exploration of the apparatus by the rats was evaluated when they raised their bodies and contact their paw(s) on the cylinder wall (20 movements are counted). The apparatus consisted of a transparent glass cylinder 20 cm in diameter and 30 cm in height. All animals were submitted to this task 2 h before surgery, to verify the basal forelimb symmetry. The CT was repeated on the 3rd, 7th, 14th, 21st, 28th, 35th and 42nd days after EVs treatment. The performance was recorded using ANY-Maze software (Stoelting CO., Wood Dale, IL), and the ipsilateral (to the lesion), contralateral or both front paws preference were counted in a blind analysis. The asymmetry of each animal was calculated by the following formula: asymmetry = (% of ipsilateral paw use = Ipsilateral paw use / sum Ipsilateral + Contralateral + use of both paws) − (% of contralateral paw use = contralateral paw use / sum of Ipsilateral + Contralateral + use of both paws). The asymmetry percentage was converted into symmetry percentage[43]. Groups: Naive (n = 6), ISC (n = 22), ISC + EV0 (n = 17); ISC + EV1 (n = 16) and ISC + EV2 (n = 17). At the end of each task, the apparatus was cleaned using 70% ethanol solution.
6b. Open Field Task (OFT)
The open field task evaluates habituation to novelty (assessing short- and long-term memory - exploratory activity) and locomotor activity in an arena[53]. The apparatus consisted of a black cage measuring 50 cm in length × 50 cm in width × 50 cm in height. The sessions lasted 10 min (individually). Animals performed the task on the 7th, 21st and 42nd days after EVs treatment. Short-term memory (habituation to novelty) was evaluated considering the decrease of locomotion during the first 5 min of the 1st session (7th day). Long-term memory was evaluated considering the decrease in locomotion during the first minute through the successive sessions (from the 1st to the 3rd session). Groups: Naive (n = 8), Naïve + EV0 (n = 7), ISC (n = 22), ISC + EV0 (n = 17); ISC + EV1 (n = 16) and ISC + EV2 (n = 17). At the end of each session, the apparatus was cleaned with 70% ethanol solution. The task was recorded and analyzed using ANY-maze 6.1 software.
6c. Novel Object Recognition Task (NORT)
The behavioral sessions lasting 10 min were performed on the 7th, 21st and 42nd days after EVs treatment. 90 min after the OFT session, Object Recognition (OR) short- and long-term memories were evaluated[54]. Animals were individually placed on the periphery of the arena for exploration. Two identic familiar objects (FOs) were placed in the arena and animals were allowed to explore them for 10 min. Sniffing and touching the objects were considered as exploratory behavior. 90 min after the training session, each animal was placed back into the arena to evaluate short-term memory. One of the 2 FOs used in the training session was replaced by a new distinct object (NO). The long-term memory was evaluated 24 h after the short-term memory task session, when the animals were placed back in the arena with same FO used in the training session and in first test session (short-term memory), while the same NO was displaced to a different position. In all sessions, the time spent exploring the objects was recorded by using ANY-maze 6.1 software. Results were expressed as a percentage of time exploring each object. Animals that recognized the novel object (short-term memory) or its new position (long-term memory), explored it more than 50% of the total exploring time of both objects. Groups: Naive (n = 8), Naive + EV0 (n = 7), ISC (n = 22), ISC + EV0 (n = 17); ISC + EV1 (n = 5) and ISC + EV2 (n = 16). At the end of each session, the apparatus was cleaned using 70% ethanol solution.
6d. Elevated Plus-maze Task (EPMT)
The EPMT task is widely used to study anxiety-like behavior[55]. The apparatus had 2 open arms (50 cm long × 10 cm wide) and 2 closed arms (50 cm long × 10 cm wide × 40 cm high), separated by a central platform (5 cm long × 5 cm wide). The apparatus was placed 70 cm high from the floor. The animals were kept in a red-light area for 1 hour before starting this task. ANY-maze software was used to record behavioral performance for 5 min. The percentage of time spent in open and closed arms was assessed. Anxiety-like behavior was considered as the increase of time spent on closed arms. Each animal conducted this once, on the 7th day after treatment with EVs. At the end of each session, the equipment was cleaned with 70% alcohol.
7. Statistical Analysis
The size of the brain lesion, BBB integrity, number of vesicles found in brain tissue and angiogenesis analysis were evaluated by unpaired T-tests. Two-way RM ANOVA was applied for CT, followed by Sidak’s multiple comparisons test. Short-term memory was evaluated by unpaired t-test. Long-term memory was evaluated by two-way ANOVA followed by Sidak's multiple comparisons test. Unpaired T-tests were used for the NORT with a theoretical average of 50%. Data are reported as the mean ± SD. All analyses were performed using Graph Pad Prism 6.0.