Isolation of MSCs from Wharton’s jelly and Confirming of the isolated MSCs
Stem cell isolation and characterization procedures were performed as described in our previous study [23]. After signing the informed consent form by patients and a witness and approving by Institutional Review Board (IRB), human umbilical cords were collected from full-term cesarean section births at Akbar-Abadi Hospital and transferred to the Cellular and Molecular Research Center at Iran University of Medical Sciences (IUMS), in a sterile specimen container containing 0.9% normal saline.
Cell culture: Briefly, after washing the cords with phosphate-buffered saline (PBS) and separating the blood vessels and the amniotic membrane, the remaining tissue known as Wharton’s Jelly was cut into small pieces of 5 mm3 and transferred into a sterile centrifuge tube containing the enzymes collagenase type I (300 U/ml) and hyaluronidase (1 mg/ml) for the first enzymatic digestion putting in an incubator (at 37oC in 5% CO2) for 1 h. After filtering the lysed solution with a 70 μm cell strainer and centrifuging at 300 g for 5 min, the remaining tissue was transferred to another centrifuge tube containing 0.1% trypsin enzyme (Sigma-Aldrich, St Louis, MO, USA) for the second enzymatic digestion for 30 min to isolate cells as much as possible. At the end of the second enzymatic digestion, it was again filtered and centrifuged. Both cell pellets derived from two enzymatic digestion steps were mixed, suspended, and cultured in a complete culture medium included Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Billings, USA) containing 10% fetal bovine serum (FBS; Sigma, Missouri, USA) and 1% penicillin-streptomycin antibiotic (Invitrogen, Waltham, USA) for expansion. The viability of the isolated cells was assessed by the trypan blue exclusion method using 0.4% trypan blue dye (Sigma-Aldrich, St Louis, MO, USA).
Flow cytometry: To quantitatively detect the mesenchymal CD markers on Wharton’s jelly-isolated cells, passage three cells were incubated with monoclonal mouse anti-human antibodies against mesenchymal markers CD105 and CD73 (positive markers) and hematopoietic markers CD45 and CD34 (negative markers) followed by 10 mg/ml of fluorescein isothiocyanate (FITC)–conjugated anti-mouse immunoglobulin G (IgG) antibodies (Abcam, Cambridge, UK) for 1 h at room temperature (RT). A FACS machine (Becton, Dickinson, Franklin Lakes, NJ, USA) and FlowJo software were used for antibody binding analysis and data analysis, respectively.
Immunocytochemistry (ICC): To qualitatively confirm mesenchymal CD markers on Wharton’s jelly-isolated cells, passage three cells were incubated with primary anti-human nuclei antibodies against mesenchymal markers CD105, CD73 (positive markers), hematopoietic marker CD31 (negative markers), and neuronal marker β-tubulin III (negative markers). The coverslips were mounted and observed under a Nikon Eclipse TE300 inverted microscope (Spectra Services, Ontario, NY, USA) and images were captured by a CCD camera connected to the microscope.
Osteogenic and Adipogenic Differentiation: Passage three cells were incubated in two differentiation mediums: i) 14 days in the osteogenic induction medium (DMEM‐LG plus 10% FBS, 50 μg/ml ascorbate‐2 phosphate, 10−8 M dexamethasone, and 10 mM β‐glycerophosphate [Invitrogen, Waltham, USA]); and ii) 21 days in the adipogenic differentiation medium (DMEM and 1g/ml glucose [DMEM‐LG] plus 10% FBS, 50 μg/ml of ascorbate‐1 phosphate, 10−7 M dexamethasone, and 50 μg/ml indomethacin [Invitrogen, Waltham, USA]). The medium in both induced cell types was changed every 3 days, and completion of cell differentiation was established by morphology and its related staining (i.e., Alizarin Red S for osteocytes and Oil Red O for adipocytes, [Sigma, Missouri, USA]).
Alzheimer’s Disease (AD) Modeling and Intranasal (IN) Administration of WJ-MSCs
All animal experiments were performed according to the guidelines of the ethical committee of IUMS (IR.iums.rec.1397.1299). Adult 220–260 gr male Wistar rats were obtained from IUMS animal lab and AD modeling was performed as described in our previous study [23]. Briefly, rats were anesthetized using ketamine/xylazine (50/4 mg/kg, i.p), and fixed in the stereotaxic device. After exposing the skull, freshly prepared amyloid β 1–42 (Sigma, 8 µg/kg of Aβ1–42 in 16 μl PBS) was administered using a hamilton microsyringe during 3 min into the dorsal hippocampus bilaterally according to Paxinos rat brain atlas (coordinates: 3.6 mm posterior, ± 2 mm lateral to the bregma, and 3.2 mm ventral to the skull surface).
The rats were randomly divided into 3 groups (n=8 in each group) as follows: i) control group (vehicle [PBS]-treated rats), ii) AD model group (Aβ-treated rats), and iii) MSC-treated group (AD models those which treated with IN administration of WJ-MSCs).
IN-administration of WJ-MSCs was performed on day 14 after AD induction as previously described [21, 24]. Briefly, animals anesthetized and immobilized facing upward. First, 100U hyaluronidase was freshly dissolved in sterile PBS (4 U/μl) and 3 μl of the suspension administered in each nostril using a pipette, which was repeated 4 times up to almost 100U of hyaluronidase suspension. Next, after keeping treated rats facing upward for 30 min, 3 ×105 WJ-MSCs/rat was suspended in 36 μl PBS and administered 6 μl/nostril. After 30 s, that the sample drops were completely disappeared the administration with a 2 min interval repeated 3 times.
Behavioral Evaluations
Two months after cell therapy, rats were evaluated to assess cognitive functions (learning and memory) performing passive avoidance (PA) response and Morris water maze (MWM) tests.
Passive Avoidance (PA) Response: This test was performed using a shuttle box device with two connected chambers of equal size separated by a guillotine door as previously described [25]. First, a habituation trial was performed to make all animals initially familiar with the environment and apparatus without any stimuli. Next, for the acquisition trial, animals of all groups were guided individually into the illuminated chamber for 10 seconds opening the guillotine door to note the latency to enter the dark chamber as initial latency (IL). After entering the dark chamber, the animals’ feet were exposed to electrical stimulation (0.5 mA, 50 Hz, 2 seconds once) through the stainless steel floor in the dark chamber. Finally, after 24 h, for the retention trial, the rats were re-entered into the lighted chamber without any foot shock to record latency to enter the dark chamber as retention time (step-through latency, STL). And total time spent of rats in the dark chamber (time spent in the dark chamber) was also recorded as an indicator of contextual learning. The maximum cut-off time for the STL and time in the dark chamber was 300 and 600 seconds, respectively. If a rat avoided entering the dark chamber for up to 300 seconds, the acquisition of PA response would be considered as successful for that rat.
Morris Water Maze (MWM): Spatial reference learning and memory were evaluated in the water maze task as previously described [26, 27]. This test was performed in 6 days including the habituation day (day 1 – apparent platform in the center of tank), acquisition phase (day 2 to 5 – hidden platform in one of the quadrants), and the probe trial stage (day 6 – no platform) using a circular water (22 °C) tank that was divided into four imaginary quadrants with a platform as previously described [28, 29]. In the acquisition phase, the learning process was conducted for 4 days and each day for 4 trials (each trial was set as 60 sec.). In the probe trial test on the sixth day, spatial memory was evaluated by removing the hidden platform. In this stage, time spent and distance traveled in the target quadrant (where the platform situated in earlier phases) were measured as two criteria of spatial memory. In both acquisition and probe trial stages, rats’ movements were recorded by the camera above the water tank and data collected by a computer equipped with water maze software for analysis.
Histological Evaluations
RNA extraction and real-time quantitative reverse transcription PCR: Total RNA from hippocampus was extracted and purified with a TRIzol™ (Sigma, Pool, UK) according to manufacturer's instructions, and its concentration was measured with a NanoDrop ND-100 spectrophotometer. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) was employed to quantify the gene expression levels of neurotrophic factors BDNF and NGF and apoptosis-related factors BCL2, BAX, and Caspase 3 in AD rat hippocampus after treatment with WJ-MSCs. 500 nanograms of RNA were reverse transcribed into complementary DNA (cDNA) with the Transcriptor High Fidelity cDNA Synthesis kit (Invitrogen, Paisley, UK) using oligo (dT) primers (Roche). 1 µl of the cDNA was amplified using Opticon II (Invitrogen, Paisley, UK) and the SYBR Green PCR Master Mix (Invitrogen, Paisley, UK), following the manufacturer's instructions. PCR was performed in 40 cycles using an annealing temperature of 60°C for all genes. Primers used in this study were specifically designed between two adjacent exons (gene runner program) and the sequences have been listed in Table2. mRNA levels for target genes were normalized to reference gene (β-actin) by subtracting the Ct (cycle threshold) value of the reference gene (β-actin) from the Ct value of the samples (ΔCt = Ct Sample-Ct reference). The relative expression of the target gene to a calibrator is quantified using 2-ΔΔCt.
Cresyl Violet (Nissl) Staining: Two months after cell therapy, this test was performed to distinguish healthy neurons from damaged neurons in the cornu ammonis-1 (CA1) area of rats’ hippocampus [30]. Briefly, the samples were fixed, 7μm coronal sections (5 sections) were taken from -3.84 to -5.8 from Bregma, the sections were transported to gelatinized slides and stained with cresyl violet stain. The images from stained slides were taken by an optical microscope (Carl Zeiss; Oberkochen, Germany) and were averaged using ImageJ software for dark cells in the CA1.
Statistical analyses
The data were normalized and analyzed using one-way ANOVA followed by Tukey post hoc test to determine the statistical significance between different groups using SPSS software version 24 (SPSS Inc., Chicago, IL, USA). Also, analysis of PA response data was analyzed by paired t-test and a significant difference between groups was determined by one-way ANOVA. All results were considered significant at P < 0.05 and expressed as mean ± SEM. Image analysis was done with ImageJ software.