Human tissues
Human fetal livers (FL) (range 7–9 weeks of gestation) were obtained from voluntary abortions (Obstetrics and Gynecology Department, Rene Dubos Hospital, Pontoise, France). Developmental age was estimated based on several anatomic criteria according to the Carnegie classification for embryonic stages74 and by ultrasonic measurements for fetal stages. Bone marrow (BM) aspirates were obtained from patients undergoing a total hip replacement surgery (HIA, Percy Hospital, Clamart, France and Orthopedic Service, Polyclinic of Blois, Blois France). Human adult peripheral blood from healthy adults was obtained from the French Establishment of Blood (EFS) (Rungis, France).
MSC isolation and culture
FL samples were excised sterilely using microsurgery instruments and a dissecting microscope, in phosphate‐buffered saline (PBS) containing 1% penicillin/streptomycin (P/S) (all from GIBCO). Tissues were dissociated for 1 hour at 37°C in α-minimum essential medium (α-MEM; GIBCO) containing 10% fetal calf serum (FCS; Eurobio), 0.1% type I/II/IV collagenase and type VIII hyaluronidase (Sigma‐Aldrich). FLs were then disrupted mechanically through 18‐, 23‐ and 26‐gauge needles successively. BM cells were extracted and disrupted mechanically from hip surgery samples, in PBS containing 1% P/S (all from GIBCO). BM and FL mononuclear cells were then isolated using a density Ficoll gradient separation (Pan-Biotech) and plated at 1.5x105 cells/cm2 in α-MEM medium supplemented with 1% GlutaMAX, 1% P/S solution (all from GIBCO), and 10% heat-inactivated FCS (Eurobio) at 37°C in 5% CO2. After three days, non-adherent cells were discarded and the medium was changed every week. When the adherent cells were 70–80% confluent, they were harvested by treating with trypsin-EDTA (GIBCO) and seeded at 4,000 per cm2 in α MEM complete medium. MSCs were amplified from passages 0 to 3. In all experiments MSCs were used from passage 3 to 6. For morphology analysis images were acquired at 4x magnification at early, mid, and late passage numbers using the EVO XL Core Imaging System from Life technologies. For immune-phenotyping analysis MSCs were recovered and stained with fluorochrome-conjugated antibodies for MSC (APC-anti CD271 (LNGFR), PE-anti-CD166 (ALCAM), APC-anti-CD146 (MCAM), FITC-anti-CD90 (Thy-1), PE-anti-CD106 (VCAM-1), PE-anti-CD105 (Endoglin), PE-anti-CD73, APC-anti-CD54 (ICAM-1), APC-anti-CD51 (Integrin αV), PE-anti-CD44 (HCAM), APC-anti-CD29 (β1-integrin), hematopoietic (FITC-anti-CD45, PE-anti-CD14, and APC-anti-CD34), endothelial (PE-anti-CD144) markers and major histocompatibility (MHC) (FITC-anti-HLA-DR, FITC-anti-HLA ABC) antigens as described below.
MSC differentiation assay
FL and BM-MSCs were tested for their ability to differentiate into adipocytes and osteocytes (R&D Systems). For osteogenic differentiation MSCs were seeded in 24-well plates at 3,000 per cm2 in α-MEM supplemented with 10% FCS and 1% P/S. After cell adhesion, medium was removed and replaced by α-MEM supplemented with 10% FCS, 1% P/S and 0.052 μg/ml dexamethasone, 12.8 μg/ml ascorbic acid, and 2.15 mg/ml β-glycerophosphate from Sigma-Aldrich. Cells were cultured for 12 days to 3 weeks at 37°C in 5% CO2 atmosphere, and medium was changed 2 times in a week. Quantification of mineralization was performed after fixation in 4% paraformaldehyde for 10 minutes and incubation in a solution of 2% Alizarin Red S (Sigma-Aldrich) for 5 minutes. Cells were then washed and dried. A buffer composed of 0.5N hydrochloric acid and 5% SDS was added for extraction of Alizarin Red S staining and read at 405 nm. For adipogenic differentiation, MSCs were seeded at 21,000 per cm2 in α-MEM medium supplemented with 10% FCS and 1% antibiotics. After cells reached confluence, medium was removed and DMEM high glucose supplemented with 10% FCS, 1% antibiotics, 0.52 μg/mL dexamethasone, 0.2 mM indomethacine, 0.01 mg/mL insulin and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (All from Sigma-Aldrich) were added for 3 days. Medium was removed and DMEM high glucose supplemented with 10% FCS, 1% antibiotics and 0.01 mg/mL insulin were added for 1 day. Medium was removed for 2 additional cycles as previously and incubated in DMEM high glucose supplemented with 10% FCS, 1% antibiotics and 0.01 mg/mL insulin for the last week. Cells were cultured for 3 weeks at 37°C in 5% CO2 atmosphere. Differentiation into adipocytes was evaluated after fixation in 4% paraformaldehyde for 10 minutes and incubation in a solution of 0.3% Oil Red O for 5 minutes.
MSC proliferation assay
FL and BM-MSC proliferation potential was evaluated using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Cat. No G3582, Promega) according to the manufacturer's instruction. The amount of soluble formazan product produced by the reduction of MTS by metabolically active cells was measured at the 490 nm absorbance using the microplate fluorometer Fluoroskan Ascent® from Thermo Fisher Scientific.
T cell isolation and culture
Murine pan T cell isolation kit II (Miltenyi Biotec) was used to isolate total CD3+ T cells from pooled spleens of 6 to 12 weeks-old WT C57BL/6 mice (Envigo and Charles River).
Human pan T cell isolation kit (Miltenyi Biotec) was used to isolate total CD3+ T cells from MNCs of peripheral adult blood. Furthermore, CD25+ cells were depleted from the mouse and the human CD3+ T cell population using either anti-mouse CD25 biotin-conjugated antibody (BD biosciences) or anti-human CD25 biotin-conjugated antibody (Miltenyi Biotec), and anti-biotin microbeads (Miltenyi Biotec). The magnetic-activated cell sorting (MACS) method was used in all cell isolation steps. The resulting murine or human CD3+CD25- T cells, ≥ 95% pure, were cultured in the presence of BM and FL-MSCs.
MSCs/T cells co-culture
5x104 BM or FL -MSCs were seeded in 6-well plates and incubated for 24h in α-MEM containing low glucose, 1% GlutaMAX, 1% P/S (all from Gibco) and 10% heat-inactivated FCS (Eurobio). Freshly isolated murine CD3+CD25-T cells were then added at different ratios to BM or FL-MSCs, depending on experimental conditions, in RPMI medium containing 1% P/S, 1% HEPES, 5x10−5 M β-mercaptoethanol (all from GIBCO) and 10% FCS (FCS; Eurobio). All co-cultures were performed in 50% α-MEM-50% RMPI complete medium at 37°C in 5% CO2. For FACS analysis, T cells were removed by a gentle cell resuspension followed by a cell aspiration (T cells stay in suspension and MSCs adhere to the plastic). In order to avoid the integration of potentially contaminating MSCs to T cell results, cells were first gated on CD4+ or CD8+ T cell markers prior analysing any further markers.
T lymphocytes proliferation assay
Freshly isolated CD3+CD25- murine or human T cells were labelled with carboxy fluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes) and stimulated by Dynabeads mouse or human T-activator CD3/CD28 respectively (Gibco) according to the supplier’s protocol. 5x104 BM or FL-MSCs seeded in 6-well plates one day before, were then co-cultured with increasing numbers of CFSE labeled, activated CD3+CD25- murine or human responder T cells (MSC to T cell ratios used: 1:1, 1:2, 1:4, 1:6, 1:8 and 1:10) in a total volume of 3ml of 50% RPMI-50% MEMα medium. 1x105 CFSE labeled, activated or non-activated murine or human CD3+CD25- T cells grown alone in 50% RPMI-50% MEMα medium were used as controls. After 3 days of co-culture, murine or human T cells were harvested by gentle aspiration and stained with Vioblue-anti-mouse CD4 and PE-Vio770-anti-mouse CD8α antibodies or Vioblue-anti-human CD4 and PE-Vio770-anti-human CD8 antibodies respectively (Miltenyi Biotec). The percentage of proliferating cells among CD4+ and CD8+ T cells was analyzed by flow cytometry measurements of the dilution of CFSE using LSRFortessa flow cytometer (BD Biosciences) and the FlowJo v10 software’s proliferation tool (FlowJo LLC). Cells undergoing division were identified by the decrease in CFSE resulting from dilution of the dye with each division. CFSE labeled, non-activated murine CD3+CD25- T cells grown alone consisted of non-proliferating cells (CFSE bright) with less than 5% CFSE dim proliferating cells.
T lymphocytes activation assay
Freshly isolated CD3+CD25- murine T cells were stimulated by Dynabeads mouse T-activator CD3/CD28 (Gibco) according to the supplier’s protocol. 5x104 BM or FL -MSCs seeded in 6-well plates one day before, were then co-cultured with 25x105 activated CD3+CD25- murine responder T cells (MSC to T cell ratio used: 1:5) in a total volume of 3ml of 50% RPMI-50% MEMα media. 1x105 activated and non-activated murine CD3+CD25- T cells grown alone in 50% RPMI-50% MEMα medium were used as controls. After either 1 or 3 days, murine T cells were harvested by gentle aspiration and stained with either VIOBLUE-anti-CD4, VioBright FITC-anti-CD8α, PE-anti-GITR, PE-Cy7-anti-CD25 (all from Miltenyi Biotec) and PE-Cy5.5-anti-FoxP3 (eBioscience) or anti-CD4-VIOBLUE, FITC-anti-CD8α, PE-Vio770-anti-ICOS, APC-anti-TNFR2 (all from Miltenyi Biotec) and PE-Cy5.5-anti-FoxP3 (eBioscience) antibodies (Abs).
Regulatory T cell induction assay
Freshly isolated CD3+CD25- murine T cells were stimulated by Dynabeads mouse T-activator CD3/CD28 (Gibco) according to the supplier’s protocol. 5x104 BM or FL -MSCs seeded in 6-well plates one day before, were then co-cultured with increasing numbers of, activated murine CD3+CD25- responder T cells (MSC to T cell ratios used: 1:1, 1:2, 1:4, 1: 6, 1:8 and 1:10) in a total volume of 3ml 50% RPMI-50% MEMα medium. 1x105 murine activated and non-activated CD3+CD25- murine T cells grown alone in culture were used as controls. After 4 days, T cells are harvested and stained using the following Abs: VIOBLUE-anti-CD4, FITC-anti-CD8α, PE-Cy7-anti-CD25, PE-anti-CTLA4, APC-anti-TNFR2 (all from Miltenyi) and Foxp3-PE-Cy5.5 (eBioscience) or anti-CD4-VIOBLUE, FITC-anti-CD8α, PE-anti-GITR, PE-Vio770-anti-ICOS (all from Miltenyi Biotec) and PE-Cy5.5-anti-FoxP3 (eBioscience).
Induced T reg suppressive capacity assay
The CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec) was used to isolate induced regulatory T cells (iT regs) following a 4 day co-culture of activated CD3+CD25- murine T cells with FL or BM -MSCs. Briefly, CD4+ T cells were first negatively selected on a separation column, then the CD4+CD25+ subset was purified using CD25 microbeads (Miltenyi Biotech) according to the manufacturer's instructions. To determine the purity of the isolated iT regs, we performed a flow cytometry analysis using VIOBLUE-anti-CD4, FITC-anti-CD8α, PE-Cy7-anti-CD25 (all from Miltenyi) and Foxp3-PE-Cy5.5 (eBioscience) antibodies combination. The purity of CD4+CD25+ iT regs was consistently higher than 90%, as confirmed by FACS. Isolated iT regs were then co-culture in 96-well round bottom plates with increasing number of freshly isolated, CSFE labeled and CD3/CD28 activated murine CD3+CD25- responder T cells using different iT reg to T cell ratios (1:1, 1:5, 1:10) in a total volume of 200 µl of RPMI medium containing 1% P/S, 1% HEPES, 5x10−5 M β-mercaptoethanol (all from GIBCO) and 10% FBS (Pansera ES; PAN-Biotech) at 37°C in 5% CO2 as previously described.75 1x105 CSFE labeled, activated and non-activated murine CD3+CD25- T cells grown alone in culture were used as controls. After 3 days of co-culture, cells were harvested and stained using VIOBLUE-anti-CD4 and FITC-anti-CD8α antibodies combination. The percentage of proliferating cells among CD4+ and CD8+ T cells was analyzed by flow cytometry measurements of the dilution of CFSE using LSRFortessa flow cytometer (BD Biosciences) and the FlowJo v10 software’s proliferation tool (FlowJo LLC). CFSE labeled, non-activated T cells cultured alone were used as non-proliferating cell control (CFSE bright) with less than 5% CFSE dim proliferating cells. CSFE negative cells corresponding to iT regs were gated out for the analysis. To ensure that only T conv cell proliferation was measured and that iT regs did not contribute to the proliferation observed wells containing only iT regs were included in all experiments.
MSC and T-cell cell analysis by flow cytometry
Antibodies used for flow cytometry analysis are listed in Supplementary Table 1.
For MSC immune-phenotyping, cells developed in culture were harvested by non-enzymatic treatment (Cell dissociation solution; Sigma-Aldrich) and washed in complete medium and resuspended in PBS (GIBCO) 0.2% bovine serum albumin (BSA) (Sigma-Aldrich). For T cells immune-phenotyping, cells were recovered by gentle aspiration, washed in complete medium and washed and re-suspended in PBS (GIBCO) 0.2% bovine serum albumin (BSA) (Sigma-Aldrich). In all cases, 105 recovered cells were incubated for 30 min on ice with fluorochrome-conjugated antibodies, washed and re-suspended in PBS (GIBCO) 0.2% bovine serum albumin (BSA) (Sigma-Aldrich), for analysis
Intracellular Foxp3 staining was performed after cell surface antibodies staining, according to the manufacturer’s instructions, using Foxp3 staining buffer set from eBioscience. Flow cytometric analysis was performed using LSRFORTESSA flow cytometer (BD Biosciences) and analyzed using FlowJo v10 software (FlowJo-LLC). Background staining was evaluated using isotype-matched control antibodies and 7-Amino-Actinomycine D (7AAD) (Sigma-Aldrich) was used to gate dead cells out.
Statistical analysis
Prism software (GraphPad) was used for statistical analysis. Student t test or one-way ANOVA with post hoc analysis was performed depending on the number of comparatives. For cytometry analysis, we have normalized the MFI values with T-cell alone control group or BM-MSCs derived iT regs for T regs activation marker experiments. Then we used unpaired, two-tailed Student t tests or one-way ANOVA for P-value generation. The data are represented as mean ± SEM. ns, non-significant; *P < .05, **P < .01, ***P < .001.Correlation coefficient is significant at 0.8 < CC < 1, P***, 0.8 < CC < 0.6 P**, 0.6 < CC < 0.4 P* and non-significant at CC > 0.4.