Isolation of MSCs from WJ and confirmation of isolated cells
Stem cell isolation and characterization procedures were performed as described in our previous study [23]. Briefly, human umbilical cords were collected from full-term cesarean section births, the blood vessels and amniotic membrane were separated, and the remaining tissue, known as WJ, was cut into small pieces of 5 mm3. Next, they were transferred into a sterile centrifuge tube containing collagenase type I (300 U/mL) and hyaluronidase (1 mg/mL) enzymes for the first enzymatic digestion in an incubator (37oC in 5% CO2) for one hour. After filtering the lysed solution with a 70-μm cell strainer and centrifuging at 300 g for five minutes, 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 minutes. Next, it was filtered and centrifuged again. Both cell pellets derived from two enzymatic digestion steps were mixed, suspended, and cultured in a complete culture medium, including Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Billings, USA), containing 10% fetal bovine serum (FBS; Sigma, Missouri, USA) and 1% penicillin-streptomycin solution (Invitrogen, Waltham, USA) for expansion. The viability of 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 in WJ-isolated cells, passage 3 cells were incubated with monoclonal mouse anti-human antibodies against mesenchymal markers (CD105 and CD73) as positive markers and hematopoietic markers (CD45 and CD34) as negative markers, followed by 10 mg/mL of fluorescein isothiocyanate (FITC)–conjugated anti-mouse immunoglobulin G (IgG) antibodies (Abcam, Cambridge, UK) for one hour at room temperature (RT). Also, a fluorescence-activated cell sorting (FACS) analyzer (Becton, Dickinson, Franklin Lakes, NJ, USA) and FlowJo software were used for antibody binding analysis and data analysis, respectively.
Immunocytochemistry (ICC): To qualitatively confirm the mesenchymal CD markers on WJ-isolated cells, passage 3 cells were incubated with primary anti-human nuclei antibodies against mesenchymal markers (CD105 and CD73), hematopoietic marker (CD31 as a negative marker), and neuronal marker (β-tubulin III as a negative marker). The coverslips were mounted and observed under a Nikon Eclipse TE300 inverted microscope (Spectra Services, Ontario, NY, USA), and images were acquired by a charge-coupled device (CCD) camera, connected to a microscope.
Osteogenic and adipogenic differentiation: The passage 3 cells were incubated in two differentiation media, that is, an osteogenic induction medium (DMEM‐LG+10% FBS, 50 μg/mL of ascorbic acid 2-phosphate, 10−8 M dexamethasone, and 10 mM β‐glycerophosphate; Invitrogen, Waltham, USA) for 14 days and an adipogenic differentiation medium (DMEM+1 g/mL of glucose [DMEM‐LG], 10% FBS, 50 μg/mL of ascorbate 1 phosphate, 10−7 M dexamethasone, and 50 μg/mL of indomethacin; Invitrogen, Waltham, USA) for 21 days. The media in both cell types were changed every three days, and completion of cell differentiation was established by morphology and staining (i.e., Alizarin Red S for osteocytes and Oil Red O for adipocytes; Sigma, Missouri, USA).
AD modeling and IN administration of WJ-MSCs
Adult 220–260 g 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 assessments
Two months after cell therapy, to evaluate the effect of WJ-MSCs on spatial learning and memory, two tests, including passive avoidance (PA) response and Morris water maze (MWM), were conducted:
PA response: This test was performed using a shuttle box device with two connected equal-sized chambers, separated by a guillotine door, as previously described [25]. First, a habituation trial was performed to familiarize all animals with the apparatus without any stimuli. Next, for the acquisition trial, the animals in all groups were guided individually into the illuminated chamber for ten seconds, and the guillotine door was opened to determine the latency to enter the dark chamber as the initial latency (IL). Once the animal entered the dark chamber, its feet were exposed to electrical stimulation (0.5 mA, 50 Hz, 2 sec) through a stainless steel floor. Finally, after 24 hours, for the retention trial, the rats re-entered the light chamber without any foot shock to record latency to enter the dark chamber as retention time (step through latency [STL]). The total time spent by the rat in the dark chamber (time spent in the dark chamber) was also recorded as an indicator of contextual learning. The maximum cutoff time for STL and the time spent 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 was considered successful.
MWM test: Spatial reference learning and memory were evaluated in the water maze task as previously described [26, 27]. This test was performed over six days using a circular water tank (22°C), which was divided into four imaginary quadrants with a platform inside and a camera above the tank, as previously described [28, 29]. The MWM test included three phases: (1) habituation phase on the first day with an apparent platform in the center of the tank; (2) acquisition phase in which the learning goals were achieved on days 2-5 with a hidden platform in one of the quadrants (each day for four trials, with each trial lasting 60 seconds); and (3) the probe trial phase on day six without a platform in which the time spent and the distance traveled in the target quadrant (the platform was situated in the acquisition phase) were measured as two criteria for spatial memory.
Histological assessments
RNA extraction and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR): Total RNA from the hippocampus was extracted and purified with a TRIzol™ reagent (Sigma, Pool, UK), according to the manufacturer's instructions, and its concentration was measured with a NanoDrop ND-100 spectrophotometer. The RT-qPCR assay was employed to quantify the gene expression levels of neurotrophic factors (BDNF and NGF) and apoptosis-related factors (BCL2, BAX, and caspase 3) in the AD rat hippocampus after treatment with WJ-MSCs. For this purpose, 500 ng of RNA was reverse-transcribed into complementary DNA (cDNA) with the Transcriptor High-Fidelity cDNA Synthesis Kit (Invitrogen, Paisley, UK), using oligo(dT) primers (Roche, Germany). Next, 1 µL of cDNA was amplified using Opticon II (Invitrogen, Paisley, UK) and SYBR Green PCR Master Mix (Invitrogen, Paisley, UK), based on the manufacturer's instructions.
The PCR assay was performed in 40 cycles at an annealing temperature of 60°C for all genes. The primers used in this study were specifically designed between two adjacent exons in the Gene Runner program, and the sequences are listed in Table 2. The mRNA levels for target genes were normalized to the reference gene (β-actin) by subtracting the cycle threshold (Ct) of the reference gene (β-actin) from the Ct value of the samples (ΔCt= Ctsample - Ctreference). The relative expression of the calibrator target gene was quantified using the 2-ΔΔCt method.
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. An optical microscope (Carl Zeiss; Oberkochen, Germany) was used to take images from stained slides.
Statistical analysis
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 level 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 using ImageJ software.