Microarray analysis to compare miRNA expression in MSCs primed or not with IFN-γ
Bone marrow-derived MSCs were isolated from 6- to 8-week-old C57BL/6J mice (Harlan Laboratories), expanded, and characterized as described previously [11]. The experiments were approved by the Animal Ethics Committee of Ospedale Policlinico San Martino and by the Italian Ministry of Health (Approval Number: 384; authorization No. 230/2016-PR). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed (Decreto Legislativo 4 marzo 2014, n. 26, legislative transposition of Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes).
Expanded MSCs were stimulated with 10 ng/ml IFN-γ for 24 hours at 37° C, as described previously, in order to increase their immunomodulatory features [23]. The whole mRNA fraction was isolated from three different batches of MSCs unprimed or primed with IFN-γ at passage 14/15, which were shown to be immunosuppressive as demonstrated by their ability to inhibit T-cell proliferation [23]. Microarray analysis was performed and analysed by LC Science (Houston, TX) according to the MIAME guidelines [24], using Student’s t-test to compare data from unprimed and IFN-γ-primed samples for each batch separately, as well as for pooled batches.
Isolation and characterization of MSC-derived exosomes
In order to increase their production of exosomes, expanded IFN-γ-primed and unprimed MSCs were stimulated for 20 minutes with 1 mM ATP (Sigma-Aldrich) at 37° C [25]. The resulting supernatant was centrifuged at 2,000 x g at 4° C for 20 minutes to eliminate cells and debris, and incubated overnight at 4° C with 0.5 volume of Total Exosome Isolation Kit (Invitrogen). After the incubation, the sample was centrifuged at 10,000 x g at 4° C for 1 hour and the pellet containing the exosomes was resuspended accordingly to the experimental needs.
For characterization by western blot analysis, 15 μg of exosome proteins were loaded on a precast polyacrylamide gel (from 4% to 12% gradient, Life Technologies), using the Bolt® Mini Gel Tank (Life Technologies) system. Protein were then transferred on a nitrocellulose membrane (BioRad) using XCell II™ Blot Module (Life Technologies). After blocking in 5% BSA in PBS/Tween 20 for 1 hour, the membrane was incubated overnight at 4° C with primary rabbit anti-ALIX (1:1000, Merck Millipore, Milan, Italy) and anti-CD9 (1:1000, BD Pharmigen) antibodies in 2% BSA in PBS/Tween 20. Membranes were incubated with secondary goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (1:5000, Merck Millipore) in 2% BSA in PBS/Tween 20 for 1 hour. Membranes were developed using the ECL Plus kit (Thermo Fisher Scientific).
For characterization by electron microscopy, exosomes collected from 7x106 MSCs were fixed in a volume of 50-100 μl of 2% paraformaldehyde, according to a published protocol [26]. 5 μl of resuspended pellet was allowed to adhere to electron microscopy grids (Formvar-Carbon) for 20 minutes at 42° C. Subsequently, the grids were washed 2 times with 100 μl PBS for 3 minutes, once with 1% glutaraldehyde for 5 minutes, and finally seven times with 100 μl of distilled water for 2 minutes each. For contrast phase microscopy, the samples were transferred to 50 μl 2% uranyl acetate (UA) solution for 5 minutes and then to 50 μl of methylcellulose (MC) and UA (9 ml MC + 1 ml UA 4%) for 10 minutes in ice. The sections were dried on a filter paper and then in the air, visualized using a FEI CM10 microscope, and acquired via a Leo912ab camera.
Culture and activation of N9 microglia line cells
The murine microglial cell line N9 (Neuro-Zone srl, Italy) was plated in 75 cm2 cell culture flasks at a concentration of 5-6x105 cells in 15 ml RPMI (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) (Lonza), 100 U/ml penicillin, and 100 μg/ml streptomycin, and maintained at 37° C and 5% CO2 in incubator. The cells were activated by exposure to 1 μg/ml LPS (Sigma-Aldrich) for 30 minutes for the immunofluorescence experiments and for 24 hours for the RT-PCR experiments, as indicated in the legends to the relevant figures.
Mice
B6SJL-TgN SOD1/G93A1Gur mice expressing a high copy number of mutant human SOD1 with a Gly to Ala substitution at position 93 (referred to thereafter as SOD1G93A mice) and B6SJL-TgN (SOD1)2Gur mice expressing wild-type human SOD1 [27] (referred to thereafter as wtSOD1 mice) were originally obtained from Jackson Laboratories (Bar Harbor, ME, USA) and bred at the animal facility of the Pharmacology and Toxicology Unit, Department of Pharmacy at the University of Genoa, Genoa, where they were kept until experiments were carried out.
All mice were housed in pathogen-free conditions with food and water ad libitum. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed (Decreto Legislativo 4 marzo 2014, n. 26, legislative transposition of Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes). The research protocol was approved by the Ethical Committee for Animal Experimentation of the University of Genoa, Italy, and the Italian Ministry of Health (Project No. 75f11.3, Authorization No.482/2017-PR).
The onset of overt clinical symptoms in our SOD1G93A mouse colony occurs at approximately day 90 [28]. Animals were sacrificed at the end stage of disease, established according to an homogeneous motor impairment severity score (extension reflex and gait impairment score: 4.5 units, at around 135 days of age) as previously described [13] and characterized by an overactivation of microglia [29].
Isolation of adult primary microglia
Primary microglia were isolated from the brain of late stage SOD1G93A and age-matched wtSOD1 mice, following the protocol of Cardona et al. [30], with minor modifications. Each brain was chopped in a Petri dish and transferred into a 15 ml Falcon tube; after centrifugation, the pellet was resuspended in 2 ml of activated papain solution (Roche) containing 0.5% 14.3 mM β-mercaptoethanol (final concentration 72 µM) for 30 minutes at 37° C in a water bath, resuspending every 10 minutes. 500 μl of RPMI containing 100 µM leupeptin (R&D Systems) were added to the suspension, which was mixed thoroughly for 2 minutes. 8 ml prewarmed Dnase solution (Sigma) (composed of RPMI containing Ca2+ and Mg2+, 25 mM HEPES and 30 µg/mL Dnase) were added to the samples and incubated for 10 minutes at 37° C. Suspensions were filtered on a 100-250 μm filter and centrifuged at 450 x g at 4° C for 5 minutes. Supernatants were aspirated and the pellets were resuspended in 7.2 ml of wash solution (RPMI and 1M HEPES); 1.2 ml of FBS was mixed with the cell suspension, followed by 3.6 ml of 100% Percoll (Sigma-Aldrich). Finally, 1 ml of 10% FBS in RPMI was layered over the cell suspension and samples were centrifuged at 800 x g at 4° C for 15 minutes without brake. Pellets were resuspended in 1 ml RPMI with 10% of FBS and cells were counted. An average of 5-6 x 106 cells was obtained per single brain and the primary microglia were further purified on CD11b (Microglia) MicroBeads (Miltenyi Biotec) according to the manufacturer’s instructions. It is notoriously difficult to obtain pure mouse microglia from adult brain and we reached an average yield of 3-5x105 microglia per single brain with a final purity of 85-90%.
Microglia exposure to IFN-γ primed MSC-derived exosomes
1x105 LPS-activated N9 cells or 2-3x105 primary microglia (higher concentration of cells was used because of the low survival of primary microglia in culture) resuspended in 1 ml RPMI were plated per well in a 24-well plate in presence or absence of IFN-γ primed MSC-derived exosomes (exosIFN-γ- MSC). The quantity of exosomes added to the cultures was equivalent to that produced by MSCs at a microglia:MSC ratio of 1:3. After 24 hours at 37° C and 5% CO2, cells were processed for RNA extraction.
RNA isolation and Real Time quantification
Total RNA was isolated from N9 cells and primary microglia using QIAzol Lysis Reagent (Qiagen) according to the manufacturer’s instructions. First strand cDNA was synthesized from 1 µg of total RNA from N9 cells or 500 ng of total RNA from primary microglia using Transcriptor First Strand cDNA synthesis kit (Roche Diagnostics, Germany), in a final volume of 20 μl.
Real Time polymerase chain reaction (RT-PCR) was performed in LightCycler 480 (Roche) in duplicate in a final volume of 20 μl containing 50 ng cDNA, 1 μl of each primer pair 20 mM (TIB Mol Biol), 10 μl of FastStart Essential DNA Green Master Mix (Roche). The amplification of the 3-phosphate dehydrogenase glyceraldehyde (GAPDH) gene as housekeeping gene was adopted to normalize expression data. Primer sequences used: tumor necrosis factor (Tnf) forward (5′-TCTTCTCATTCCTGCTTGTGG-3′) and reverse (5′-GGTCTGGGCCATAGAACTGA-3′); interleukin 1b (Il1b) forward (5’-AGTTGACGGACCCCAAAAG-3′) and reverse (5’-TTTGAAGCTGGATGCTCTCAT-3’); IL-18 (Il18) forward (5’- CAAACCTTCCAAATCACTTCCT-3’) and reverse (5’- TCCTTGAAGTTGACGCAAGA-3’); Cx3cr1 forward (5′-AAGTTCCCTTCCCATCTGCT-3′) and reverse (5′- CAAAATTCTCTAGATCCAGTTCAGG-3′); nuclear receptor subfamily 4 group A (Nr4a2) forward (5′-TCAGAGCCCACGTCGATT-3′) and reverse (5′-TAGTCAGGGTTTGCCTGGAA-3′); cluster of differentiation 206 (Cd206) forward (5′-CCACAGCATTGAGGAGTTTG-3′) and reverse (5′-ACAGCTCATCATTTGGCTCA-3′); mitogen-activated protein kinase (MAPK) kinase kinase 8 (Map3k8) forward (5’-TTCCAGTGCTCATGTACTCCA-3’) and reverse (5’-GGACTGCTGAACTCTGTTTGC-3’); MAPK-activated protein kinase 2 (Mk2) forward (5’-AGTGCAGCTCCACCTCTCTG-3’) and reverse (5’-CAGCAAAAATTCGCCCTAAA-3’); GAPDH forward (5’-ATGGTGAAGGTCGGTGTGA-3’) and reverse (5’-AATCTCCACTTTGCCACTGC-3’).
For miRNA amplification, RNA was isolated from exosomes using miRNeasy Mini Kit (Qiagen) according to the manifacturer’s instructions. The cDNA was obtained from 200 ng of total mRNA using miScript II RT Kit (Qiagen). miRNA amplification was performed in LightCycler 480 (Roche) in duplicate in a final volume of 25 μl containing 2.5 ng cDNA (miScript SYBR green PCR kit, Qiagen). Amplification of Scarna-17 (Qiagen) miRNA was used to normalize expression data. Primer sequences used: miR-467f 5′-ATATACACACACACACCTACA-3′; miR-466q 5′-GTGCACACACACACATACGT-3′; miR-466m-5p 5’-TGTGTGCATGTGCATGTGTGTAT-3’; miR-466i-3p 5’-ATACACACACACATACACACTA-3’; miR-466i-5p 5’-TGTGTGTGTGTGTGTGTGTG-3’; miR-467g 5’-TATACATACACACACATATAT-3’; miR-3082-5p 5’-GACAGAGTGTGTGTGTCTGTGT-3’; miR-5126 5’-GCGGGCGGGGCCGGGGGCGGGG-3’; miR-669c-3p 5’-TACACACACACACACAAGTAAA-3’.
N9 and primary microglia transfection
1x105 cells were plated in 24-well plates in 500 μl RPMI and transfected using the HiPerFect® Transfection Reagent (Qiagen), according to the manufacturer’s instructions, with mimics specific for miRNA (miRNA Mimic miRNA, Qiagen) and with MISSION miRNA Mimic Negative Control (Sigma-Aldrich), a synthetic miRNA which does not recognize any mRNA target in cells (Cneg), and with iBONi siRNA positive control-P4M (Riboxx), which inhibits the translation of GAPDH in cells, as indicator of efficient transfection (Cpos). The sequence of mimics used are: miR-467f 5’-AUAUACACACACACACCUACA-3’; miR-466q 5’-GUGCACACACACACAUACGU-3’; miR-466m-5p 5’-UGUGUGCAUGUGCAUGUGUGUAU-3’; miR-466i-3p 5’-AUACACACACACAUACACACUA-3’; miR-466i-5p 5’-UGUGUGUGUGUGUGUGUGUG-3’; miR-467g 5’-UAUACAUACACACACAUAUAU-3’; miR-3082-5p 5’-GACAGAGUGUGUGUGUCUGUGU-3’; miR-5126 5’-GCGGGCGGGGCCGGGGGCGGGG-3’; miR-669c-3p 5’-UACACACACACACACAAGUAAA-3’.
Bioinformatics analysis of miRNA targets
Online software miRWalk 2.0 was consulted to predict specific target genes of relevant miRNAs in common among different databases, such as MicroT4, miRanda and Targetscan. Pathways which selected miRNAs might modulate, were predicted in-silico using Kyoto encyclopedia of genes and genomes (KEGG) Pathway database which predicts possible pathways based on the involvement of the miRNA itself in regulating the pathway, and Panther Classification System, which predicts the pathways in which components coded for by the predicted target genes of the miRNA are involved.
Quantification of phospho-p38 MAPK by immunofluorescence
1x105 N9 cells were seeded in glass coverslips in a 24-well plate with 500 µl RPMI + 10% FBS and incubated at 37° C and 5% CO2 for 1 hour. They were transfected for 24 hours with each miRNA individually or as a mix, and with Cneg or Cpos, and stimulated with 1 µg/ml LPS for 30 minutes. Then, cells were fixed with 350 µl PFA 4% for 20 minutes at 4° C. After three washes with 500 μl PBS, the N9 cellular membrane was permeabilized with 200 μl PBS + 0.25% Triton X-100 for 10 minutes at room temperature. After three washes with 350 μl PBS, 250 µl PBS containing 1% BSA (PBS/BSA) were added to the wells for 30 minutes at room temperature, for blocking non-specific bonds. After removing the medium, primary monoclonal rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (clone D3F9) XP® (Cell Signaling Technology; 1:2000) and mouse anti-GAPDH antibody (Sigma-Aldrich; 1:1000) in 200 µl PBS/BSA were added per well and the cells were incubated at room temperature for 1 hour. After three washes with 350 μl PBS, N9 cells were incubated with cross-absorbed secondary antibodies, Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) (Invitrogen; 1:1000) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Invitrogen; 1:3000) in 100 µl PBS/BSA for 45 minutes at room temperature in the dark. After three washes with 350 μl PBS, cells were exposed to DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) (Invitrogen) for 2 minutes and washed twice with 100 µl PBS. Coverslips were fixed with Fluoromount™ Aqueous Mounting Medium (Sigma-Aldrich). Fluorescence image acquisition was performed by a Leica TCS SP5 laser-scanning confocal microscope, through a plan-apochromatic oil immersion objective 63X/1.4 NA. The quantitative estimation of co-localized proteins was performed by calculating the ‘co-localization coefficients’[31].
According to Costes et al. [32], the correlation between the green and red channels was evaluated with a significance level > 95%. Costes’ approach was carried out by macro routines (WCIF Colocalization Plugins, Wright Cell Imaging Facility, Toronto Western Research Institute, Canada) integrated as plugins in the ImageJ 1.52q software (Wayne Rasband, NIH, USA).
Statistical analysis
The results are presented as mean ± standard error (SEM). Statistical analysis was performed on independent experiments using Student's t-test through the Prism 5 program (GraphPad Software, La Jolla, CA). In all analyses, P<0.05 is considered statistically significant.