Animals
Female BALB/c mice (spleen cell isolation) at the age of 8–12 weeks and male BALB/c mice (SC isolation) at the age of 3 weeks were obtained from the breeding unit of the Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.
The present study was approved by the Animal Ethics Committee of Charles University, and all experimental procedures were performed following the guidelines for the care and use of laboratory animals.
Isolation of Adipose-Derived MSCs
Adipose-derived MSCs were isolated from inguinal fat pads of BALB/c mice as we have described [27], cultured in Dulbecco's modified Eagle medium (DMEM, PAA Laboratories, Pasching, Austria) supplemented with 10% FBS (Sigma-Aldrich Corporation, St. Louis, MO, USA), antibiotics (100 mg/ml of streptomycin, 100 U/ml of penicillin) and 10 mM Hepes buffer, and maintained in culture as adherent monolayers. Cells between passages 3 and 5 were used in the experiments.
Isolation of Sertoli Cells
Briefly, testes were decapsulated with tweezers and digested with Collagenase II and DNase I in PBS, 20 min in shaking bath (32°C), centrifuged (10 min, 800g) and filtered through 70 µm and then through 40 µm cell strainer. Cells on the 40 µm cell strainer were washed out by centrifugation (10 min, 800g). The cell suspension was plated on DSA (lectin from Datura Stramonium, Sigma-Aldrich) coated flask, washed 1 h after plating with warm DMEM medium and cultured in a complete DMEM medium supplemented with glutamine, LIF (0.1 ng/ml, Peprotech Rocky Hill, NJ, USA) and FSH (0.5 ng/ml, Sigma-Aldrich) for 3 weeks with regular exchange of medium, then passaged twice a week. Cells were maintained in culture as adherent monolayers, and between passages 3–6 were used in the experiments.
Characterization of Surface Markers by Flow Cytometry
SCs and MSCs were harvested between passages 3–5 and washed with PBS/0.5% BSA and incubated for 30 min on ice with FITC-labeled monoclonal antibody (mAb) anti-CD90.2 (clone 30-H12, SONY), PE-labeled mAb anti-CD105 (clone MJ7/18, BioLegend San Diego, CA, USA), PE-labeled mAb anti-CD73 (clone eBioTY/11.8, eBioscience, San Diego, CA, USA), FITC-labeled mAb anti-CD44 (clone IM7, BioLegend), FITC-labeled mAb anti-CD45 (clone 30-F11, BioLegend), FITC-labeled mAb anti-CD11b (clone M1/70, BioLgenend), PE-labeled mAb anti-CD34 (clone HM34, BioLegend), PE-labeled mAb anti-CD31 (clone 390, BioLegend). A total of 40 000 cells were analyzed after the exclusion of dead cells and debris.
Macrophages were prepared by washing the peritoneal cavity of unstimulated BALB/c mice as we described elsewhere [28], and cells (5 × 105 cells/ml) were plated in 24-well tissue culture plates (Nunc, Roskilde, Denmark) in a volume of 1 ml of RPMI-1640 medium supplemented with 10% FBS (Sigma-Aldrich), antibiotics (100 mg/ml of streptomycin, 100 U/ml of penicillin) and 10 mM Hepes buffer (referred as complete RPMI-1640 medium), for 48 h in the presence or absence of SCs or MSCs (peritoneal cells: SC/MCs at a ratio 1:5, 1:10 or 1:20). To determine the phenotype of macrophages, cells were harvested, washed with PBS/0.5%BSA and incubated for 30 min on ice with Alexa Fluor 700-labeled mAb anti-CD45 (clone 30-F11, BioLegend), PE-labeled mAb anti-F4//80 (clone BM8, BioLegend), FITC-labeled aAb anti-CD206 (clone C068c2, BioLegend). A total of 50 000 cells were analyzed after exclusion of dead cells and debris. Representative flow cytometry dot plots illustrating the gating strategy are shown in Supplementary Figure S1.
In all experiments, dead cells were stained with Hoechst 33258 fluorescent dye (Sigma-Aldrich). Data were collected using LSR II cytometer (BD Bioscience Franklin Lakes, NJ, USA) and analyzed using GateLogic 400.2A software (Invai, Mentone, Australia).
RT-PCR
Total RNA was isolated from cultured SCs and murine testicular cell suspension using E.Z.N.A.® Total RNA Kit I (Omega Bio-Tek, Georgia, GA, USA) according to manufacturer's instructions, including in-column DNase treatment. Reverse transcription was performed by the SuperScript™ Reverse Transcriptase (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's instructions. Primer sequences are listed in Table S1.
Osteogenic, Chondrogenic and Adipogenic Differentiation of SCs and MSCs
SCs or MSCs were cultivated in DMEM medium to ensure mid-log growth phase confluence (60–80%). Then cells were gently harvested, and depending on the type of differentiation, were seeded into multi-well plates or Petri dishes. Cells underwent osteogenic differentiation using StemPro® Osteogenesis Differentiation Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. Osteocytes were stained with 2% Alizarin Red S solution (Sigma-Aldrich) for 15 min. A micromass culture was generated from SCs or MSCs and cultured in media prepared from StemPro® Chondrogenesis Differentiation Kit (Thermo Fisher Scientific) to induce chondrogenic differentiation. Droplets with a volume of 5 µl of the cell suspension were seeded in the centre of the 6-well plate well to generate the micromass. After incubation for 2 h under high humidity conditions, chondrogenesis media was added to the cultures. Chondrocytes were detected using 1% Alcian Blue solution on day 21. Adipogenesis was induced using StemPro® Adipogenesis Differentiation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Expanded SCs or MSCs were seed into culture flasks and cultured in an adipogenesis differentiation medium. Lipid droplets were detected on day eight by Oil Red O (Sigma-Aldrich) staining. All samples were evaluated under a light microscope.
Detection of Apoptosis
Spleen cells (1 × 106 cells/ml) were cultured in a volume of 1 ml complete RPMI 1640 medium in 24-well tissue culture plates stimulated with Concanavalin A (ConA, 1.25 µg/ml, Sigma-Aldrich) for 48 h in the presence or absence of SCs or MSCs (SCs/MSCs: spleen cells ratio 1:10 or 1:20). Cells were harvested, washed with PBS/0.5% BSA and incubated for 30 min on ice with Alexa Fluor 700-labeled mAb anti-CD45 (clone 30-F11, BioLegend), FITC-labeled mAb anti-CD4 (clone GK1.5, BD Pharmingen, San Jose, CA, USA). After washing with PBS/0.5% BSA, cells were stained for Annexin V using Annexin V detection kit according to the manufacturer's protocol (Apronex, Jesenice, Czech Republic). Dead cells were excluded using Hoechst 33258 (Sigma-Aldrich), added 15 min before flow cytometry analysis. Data were collected using LSR II cytometer (BD Bioscience) and analyzed using GateLogic 400.2A software (Invai). Representative dot plots illustrating the gating strategy are shown in Supplementary Figure S2, gated as Ki67.
Intracellular Detection of Transcription Factors
Spleen cells (1 × 106 cells/ml) were cultured in a volume of 1 ml of complete RPMI 1640 medium in 24-well tissue culture plates stimulated with ConA, (1.25 µg/ml) for 72 h in the presence or absence of SCs or MSCs (SCs/MSCs:Spleen cells ratio was 1:10 or 1:20). Cells were harvested, washed with PBS/0.5% BSA and incubated for 30 min on ice with Alexa Fluor 700-labeled mAb anti-CD45 (clone 30-F11, BioLegend), FITC-labeled mAb anti-CD4 (clone GK1.5, BD Pharmingen) and Live/Dead Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific) for staining of dead cells. Cells were then fixed and permeabilized using a Foxp3 Staining Buffer Set (eBioscience) according to manufacturer's instructions, before staining for 30 min with APC-labeled mAb anti-Foxp3 (clone FJK-16s, eBioscience), PE- labeled mAb anti-RORγt (clone AFKJS-9, eBioscience) or PE-labeled mAb anti-Ki67 (clone 16A8, BioLegend). A total of 40 000 cells were analyzed after exclusion of dead cells and debris. Data were collected using LSR II cytometerBD Bioscience) and analyzed using GateLogic 400.2A software (Invai). Gating strategy is shown in Supplementary Figure S2.
Intracellular Detection of Cytokines
Spleen cells (1 × 106 cells/ml) were cultured in a volume of 1 ml of complete RPMI 1640 medium in 24-well tissue culture plates stimulated with ConA (1.25 µg/ml) for 48 h in the presence or absence of SCs or MSCs (SCs/MSCs:spleen cells ratio 1:10 or 1:20). To analyze intracellular cytokine production, Phorbol 12-Myristate 13-Acetate (PMA, 20 ng/ml, Sigma-Aldrich), Ionomycin (500 ng/ml, Sigma-Aldrich), Brefeldin A (5 µg/ml, eBioscience) were added to the cultures for at least 4.5 h of the 48 h incubation period. Cells were harvested, washed with PBS/0.5% BSA and incubated for 30 min on ice with Alexa Fluor 700-labeled mAb anti-CD45 (clone 30-F11, BioLegend), FITC-labeled mAb anti-CD4 (clone GK1.5, BD Pharmingen) and Live/Dead Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific) for staining of dead cells. Cells were then fixed and permeabilized using a Fixation and Permeabilization Kit (eBioscience) according to the manufacturer's instructions. The cells were intracellularly stained for 30 min with PE-labeled mAb anti-TNFα (clone MP6-XT22, eBiosciece), APC-labeled mAb anti-IL-2 (clone JES6-5H4, eBioscience), APC-labeled mAb anti-IL-17A (clone eBio17B7, eBioscience). A total of 40 000 cells were analyzed after exclusion of dead cells and debris. Data were collected using LSR II cytometer (BD Bioscience) and analyzed using GateLogic 400.2A software (Invai). Representativedot plots illustrating the gating strategy are shown in Supplementary Figure S3.
Mitochondrial Transfer from SCs and MSCs to Immune Cells
Spleen cells (1 × 106 per well) were cultured in a volume of 1 ml of complete RPMI 1640 medium in 24-well tissue culture plates. SCs and MSCs were stained for mitochondria using MitoTracker® Red CMXRos (MiTT, Thermo Fisher Scientific), according to the manufacturer's instruction at the concentration 100 nM for 30 min. Stained SCs or MSCs were added to spleen cells in the ratio 1:20 (SCs/MSCs: spleen cells) and cultured together for 3 h. Cells were harvested after co-cultivation, washed with PBS/0.5% BSA and incubated for 30 min on ice with Alexa Fluor 700-labeled mAb anti-CD45 (clone 30-F11, BioLegend), and Hoechst 33258 was used for dead cells staining and exclusion. Mitochondrial transfer from SCs or MSCs to immune cells was determined as a MiTT positive population gated on CD45+ to exclude SCs and MSCs. A total number of 60 000 cells were analyzed after exclusion of dead cells and debris. Data were collected using LSR II cytometer (BD Bioscience) and analyzed using GateLogic 400.2A software (Invai).
Immunostaining of Co-cultures of Spleen Cells with SCs or MSCs – Mitochondrial Transfer Visualization
Spleen cells (1 × 105 per well) were cultured in a volume of 2 ml of complete DMEM medium in 29 mm Glass bottom dishes (Cellvis, Sunnyvale, California, USA) together with SCs and MSCs stained for mitochondria using MiTT, in ratio 1:20 (SCs/MSCs: spleen cells) for 3 h. Cell suspensions were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized by 0.2% Triton x-100 in PBS, blocked by 1% BSA in PBS and stained for 1 h with rat anti-mouse CD45 primary antibody (1:200, BioLegend) and Phalloidin Green (Sigma-Aldrich) and goat anti-rat IgG (H + L) secondary antibody Alexa Fluor 647 (Thermo Fisher Scientific) for 2 h. Staining for nuclei and sample mounting was performed by Mowiol/DAPI (Sigma-Aldrich) and observed with LeicaDmi8 fluorescence microscope.
Statistical Analysis
For the statistical analysis, the program The Prism (GraphPad Software, San Diego, CA, USA) was used. The results are expressed as the mean ± standard error (SE). The statistical significance of differences between individual groups was calculated using one-way analysis of variance (ANOVA) and Tukey post hoc test. P values less than 0.05 were considered statistically significant.