Isolation and culture of hADSCs
Adipose tissue was obtained from female donors aged 20 to 40 years in Al-Zahra Hospital of Isfahan with informed personal consent and all cell culture experiments involving human ADSCs were approved by Ethics-Committee of Ferdowsi University of Mashhad (IR.UM.REC.1400.088) and carried out in accordance with the approved guidelines. The samples were certified by the surgery room in a sterile container and brought to the laboratory on ice. Then, the following procedure was carried out:
To remove impurities and blood, the adipose tissue was washed several times with phosphate buffer saline (PBS) containing 1% penicillin /streptomycin. After chopping into smaller pieces, the samples were digested with 0.075% collagenase type I (Sigma Aldrich, Germany) prepared in PBS for 30 min at 37° C.
After 30 minutes, DMEM with 10% FBS was added to the suspension (1:1 ratio) to neutralize the enzyme. The sample tube was centrifuged for 5 minutes at 1500 rpm and stromal vascular fraction (SVF) were cultured in 75 cm2 flasks with DMEM ,10%FBS and 1% penicillin / streptomycin solution (in a 37 ° C wet incubator with 5% CO2). 4-5 days after the cells culture, the confluence of adherent cells was 80% (passage 0). For passage, cells were separated from the flask using 0.25% trypsin / 0.02% EDTA (Gibco) and transduced to new flasks (1: 3 ratio).20
Construction and production lentiviral vector and hADSCs transduction
In our previous work21 lentiviral vector based on HIV-1 that green fluorescent protein (GFP) and IFNβ, human LIF (PCDH 513B 1) gene encoders, as well as vectors (ps PAX2.2) were used as packaging vector (pMD2G) and as Envelope vector. DNA plasmids for new-combined vectors, packing vector, envelope vector and transfer vectors, were jointly transfected in HEK293 cell line.
Transduced hADSCs for GFP expression were examined by fluorescence microscopy.
Transduced hADSCs were introduced with vectors containing puromycin resistance genes and GFP gene but no IFNβ and LIF genes as control cells (GFP hADSCs).
Transduced hADSCs characterization
To confirm the mesenchymal nature of transduced hADSCs, flow-cytometry method was carried out for the analysis of specific surface markers expression (CD markers), and multilineage differentiation capacity was assessed by induction into adipocyte and osteocytes. To investigate the phenotype, the cells from passage 3-5 were incubated by non-human CD45 PerCP, CD73 FITC and CD105 PE antibodies (all from BioLegend, San Diego, California, USA) at 4 ° C for 30 minutes and then, the panel of surface markers were identified by flow cytometry (Becton-Dickinson, San Jose, CA). Each tube was evaluated 10000 times and all the data were analyzed by Flowjo software (Treestar Inc, USA).
To determine the differentiation potential of transduced hADSCs into adipocytes, 1×103 cells/cm2 of stem cells were cultured in 6-well plates. The culture medium was then switched to lipid differentiation medium (ready to use) (AdipoPlus, BI 1101). The differentiation medium was refreshed every 3 days. After 21 days, the cells were fixed in PFA 4% and incubated in 0.5% Oil Red O solution in isopropanol (Sigma) for 15 minutes at room temperature. For osteogenic differentiation, the cells were cultured in the osteogenic induction medium containing ascorbic acid (50μg / ml), sodium glycerophosphate (10 mm) and dexamethasone (10 mm) (Sigma Aldrich). After 4 weeks, the plates were washed with PBS, fixed with 4% PFA and then mineralization was confirmed by Alizarin red S staining (2%, pH = 4.1-4.3).22
EAE induction and treatment protocols
C57Bl/6 female mice (6-8 weeks old) were bought from Royan Institue of Isfahan. The mice were kept in a controlled condition (Temperature 22-26 ° C, humidity 30 40%, natural dry diet) approved by the Animal Ethics Committee for the University of Isfahan. Mice were immuned with 200 μl of MOG35-55 (400 μg) (Prospec) / CFA (Freund, Sigma Complete Supplement) (0.5 mg) (volume ratio 1: 1) emulsion injected subcutaneously into both flanks. Mice were given a total dose of 500 ng of Bordetella pertussis toxin (Sigma) on days 0 and 2 after immunization23 (all injections were given through the tail vein) and then randomly divided into the following groups (5 subjects).
1. Control (healthy group), 2. PBS (EAE group), 3. hADSCs (Un-transduced), 4. GFP- hADSCs, and 5. IFN-β/LIF-hADSCs.
On the day 12 of post immunization (DPI), early signs of motor impairment were observed, which according to previous studies, the onset of clinical symptoms is due to the inflammatory phase in the EAE model. Therefore, 12DPI was selected for the day of treatment. Prior to injection, un-transduced hADSC (passage 3-5) were labeled with PKH26 (Sigma) according to the manufacturer's protocol. For cell transplantation, 1×106 cells from each non-transduced and transduced hADSCs were resuspended in 100μl PBS on day 12DPI and injected intravenously to each mouse. For PBS group100µL, PBS was injected intravenously to each mouse on day 12 DPI. Body weight and clinical signs of all mice up to 32 DPI were recorded daily. Clinical signs were scored with: 0, without clinical signs; 1, loss of some tail tonic; 2, complete loss of tail tonicity; 3, loose tail and abnormal gait; 4, hind limb paralysis; 5, paralysis of the hind limbs with hind body passivity; 6, hind- and forelimb paralysis; and 7, death24.
Histological assessment
The spinal cord of mice was quickly dissected out after transcardial perfusion (initially with PBS, and then by 4% paraformaldehyde (PFA) mixed with PBS [pH 7.4])) at the time of killing (32 DPI). Tissues were fixed with neutral formalin 10%, embedded in paraffin and serial 5μm cross-sections were prepared. To investigate the rate of spinal cord inflammation and myelin loss, three sections of each spinal cord area (cervical, thoracic, and lumbar) were stained with hematoxylin and eosin (HE) and three sections with Luxol Fast Blue (LFB) for each animal. The sections were analyzed by a bland experimenter for the experimental groups. The inflammation score was determined as follows. 0,without inflammation; 1,scattered inflammatory cells; 2,infiltration of inflammatory cells around blood vessels; 3, significant vascular cuff with expansion in adjacent parenchyma25. Percentage of demyelinating area to the whole spinal white matter area were calculated with using NIH ImageJ software (http://rsb.info.nih.gov/ij). The following formula is used for the percentage of demyelination amount26:
The amount of demyelination (%) = (demyelination zone in white matter) / (total white matter area) × 100%
Immunofluorescence staining
The immunofluorescence was used to detect the expression of oligodendrocyte markers (Olig2 and Myelin Basic Protein [MBP]). Briefly, after re-dewatering the slides in PBS, permeability was performed by 0.1% Triton X 100 for 1 h at room temperature. Then, the slides were blocked with 3% bovine serum albumin in PBS for 30 min. The sections were incubated with primary antibodies such as: Rabbit polyclonal Anti-Olig2 (1:1000) (ab136253, Abcam, Cambridge City, MA, USA) and goat polyclonal Anti-MBP (1:200) (sc-13914; Santa Cruz Biotechnology, Inc., Santa Cruz), CA) overnight at 4 °C in a humidified chamber. After washing 3 times with PBS at room temperature, the sections were incubated with highly cross-adsorbed conjugated secondary antibodies, such as: Goat Anti-Rabbit FITC (1:1000) (ab6717; Abcam، Cambridge City، MA، USA)، Goat Anti-Rabbit Phycoerythrin (1: 1000) (ab72465; Abcam، Cambridge City، MA, USA)، Donkey Anti-Goat IgG FITC (1: 1000) (ab6881; Abcam، Cambridge City، MA، USA)، Donkey Anti-Goat Phycoerythrin (1: 1،000) (ab7004; Abcam، Cambridge City، MA، USA) for an hour at room temperature. Finally, after washing 3 times with PBS, the tissues were incubated for 5 minutes with diamidino phenylindole (DAPI) and were seen with fluorescence microscope (Olympus, BX51, Japan). Quantitative analysis of positive cells in three vision fields per section (200 / 400X magnification) was performed by ImageJ and the numbers of Olig2 and MBP-labeled cells were counted. Moreover, three mice from each group were randomly selected.20
The analysis of gene expression in CNS
To determine the expression of inflammatory and anti-inflammatory cytokines, the spinal cord was removed from the experimental groups at 32 DPI. Complete RNA was isolated from tissue using BioFACT ™ Total RNA Prep Kit and RNA concentration was determined by NanoDrop spectrophotometer. cDNA synthesis was obtained from 1μg of total RNA with cDNA reverse transcription kit (Bio FACT). The purity of the obtained cDNA was controlled by NanoDrop. To detect the expression of five genes like interlukin 17a (IL-17a), tumor necrosis factor α (TNF α), interferon γ (IFN γ) (as inflammatory cytokines) and IL 4 and IL 10 (anti-inflammatory cytokines), qRT-PCR was performed in duplicate using SYBR® Green PCR Master Mix (BioFACT) containing 5μl SYBR Green, 1μl of cDNA, 0.25μl of primer (forward and reverse mixture) and 3.75μl of nuclease-free water. Β-actin was determined as a control gene for this study. All genes were calculated using the relative quantification method (2 -∆∆CT) according to the standard protocol and mRNA expression was considered equal to the control for analysis.27 The specific primers of the above genes were designed in our laboratory by Allele ID software, version 7.0 and their sequences are listed in Table 1.
Table 1. The sequences of the applied primers in qRT-PCR
Primer name
|
Sequence (5’→3’)
|
Accession number
|
Size
|
IL-4
|
Forward : 5’-AGTTGTCATCCTGCTCTTCTT-3’
Reverse : 5’- TGTGGTGTTCTTCGTTGCT-3’
|
NM_021283.2
|
169
|
IL-10
|
Forward : 5’- GCTATGCTGCCTGCTCTT-3’
Reverse : 5’- CAACCCAAGTAACCCTTAAAGT-3’
|
NM_010548.2
|
222
|
IL-17a
|
Forward : 5’- GACTCTCCACCGCAATGA-3’
Reverse : 5’- ACACCCACCAGCATCTTC-3’
|
NM_010552.3
|
204
|
IFN-γ
|
Forward : 5’- AAAGAGATAATCTGGCTCTGC-3’
Reverse : 5’- GCTCTGAGACAATGAACGCT-3’
|
NM_008337.4
|
229
|
TNF-α
|
Forward : 5’- GTGGAACTGGCAGAAGAG-3’
Reverse : 5’- TTGAGAAGATGATCTGAGTGT-3’
|
NM_013693.3 M
|
228
|
β -actin
|
Forward : 5’- GGCTGTATTCCCCTCCATCG-3’
Reverse : 5’- CCAGTTGGTAACAATGCCATGT-3’
|
NM_007393.5 M
|
154
|
Statistical analysis
Data were reported as Mean±SEM and analyzed using SPSS version 23 (SPSS Inc, Chicago, IL, USA) and GraphPad Prism software (version 8.01). Differences in body weight and clinical score variables were assessed using repeated measures test followed by comparison of Bonferroni and other variables were analyzed using one-way analysis of variance (ANOVA) followed by Tukey post hoc test. P value <0.05 was considered as the minimum level of significant difference.
Ethics declarations
Ethical approval for this study was approved Ethics-Committee of Ferdowsi University of Mashhad (IR.UM.REC.1400.088). All experiments were performed in accordance with relevant guidelines and regulations.
This study followed the recommendations in the ARRIVE guidelines.