Dexamethasone priming enhances stemness and immunomodulatory property of tissue-specific human mesenchymal stem cells

Isolation and expansion of tissue-specific hMSCs from bone marrow, adipose tissue, dental pulp, and umbilical cord tissue

The study was approved by the Institutional Committee for Stem Cell Research (ICSCR), All India Institute of Medical Sciences (AIIMS), New Delhi, India (Ref No. ICSCR/54/16(R)). All the samples were obtained after taking the donor’s informed consent.

Bone marrow was collected from the donor undergoing the routine medical test procedure in the Department of Hematology, AIIMS, New Delhi. Briefly, BM-MSCs were isolated and cultured as previously described [23, 24]. For Adipose tissue, the sample was collected from the patients undergoing a pre-scheduled surgical procedure in the Department of Pediatric Surgery, AIIMS, New Delhi. The sample was collected in a 5 ml transport vial containing Dulbecco's Modified Eagle Medium (DMEM)-Low Glucose (LG) without FBS with 1% Penicillin (100 U/ml), Streptomycin (100 µg/ml) + Gentamycin (250 µg/ml). The Sample was washed extensively with 1X PBS containing 1% Penicillin (100 U/ml) + Streptomycin (100 µg/ml) + Gentamycin (250 µg/ml) (Gibco, USA). Then explants (~ 2 mm) were carefully placed in a 35 mm culture dish and kept undisturbed, incubated overnight at 37 °C and 5% CO2. The next day, as the tissue got adhered to the surface, complete media was added and the medium changed every 3–4 days. When cells started growing and migrating out of the explant and reached 80% confluence, cells were harvested using 0.05% trypsin–EDTA (Invitrogen-Gibco, USA) and transferred into a 60 mm culture dish. Dental pulp derived hMSCs were obtained from the third molar of each individual (16–18 years) who came for orthodontic treatment at the Department of Orthodontics and Dento-Facial Deformities, Centre for Dental Education and Research (CDER), AIIMS, New Delhi. Briefly, DP-MSCs were isolated and cultured as previously described protocol [24, 25].

Umbilical Cord derived MSCs were collected and processed within 24 h of normal or cesarean delivery from the Department of Obstetrics and Gynecology, AIIMS, New Delhi. Briefly, Umbilical Cord was collected in a 50 ml Schott bottle containing 1XPBS with 1% Antibiotics (Penicillin, Streptomycin, and Gentamycin). Upon the arrival of the sample, it was washed extensively with 1XPBS containing 1% Antibiotics. The artery part of the cord was exposed using the sharp surgical blade and it was chopped into a small piece (approx. ~ 2 mm) the exposed jelly part of the cord was placed in a 35 mm culture dish and kept undisturbed. The cultures were incubated overnight at 37 °C and 5% CO2 with 1 ml complete medium and changed every three to four days. When cells started growing and migrating out of the explant and reached 80% confluence, cells were harvested using 0.05% trypsin–EDTA (Invitrogen-Gibco, USA) and transferred into a 60 mm culture dish.

Cultures were monitored by phase-contrast microscopy (Olympus, Central Vally, PA) to evaluate the cell morphology and confluency. All assays were performed using tissue-specific hMSCs at passage 3, after their immunophenotypic characterization.

Pre-conditioning of tissue-specific hMSCs with dex

Stock concentration of Dex (Cat.No. D1756, Sigma, USA) was prepared as per the manufactures protocol. Further, the working concentration was directly prepared in the cell culture medium. Harvested cells were incubated in serum-free medium (control) or in serum-free medium containing various doses of Dex (1000 ng/ml, 2000 ng/ml, 3000 ng/ml) for assessing the effect of these drugs on immunomodulatory molecules (PGE-2, IDO and HLA-G) and stemness markers (Sox-2, Oct-4, Klf-4, Nanog, hTERT) expressed by tissue specific hMSCs. The cells were collected after different exposure times (24 h and 48 h) at all three drug concentrations of Dex. Real-time PCR, Immunofluorescence (IF) staining, Flow cytometry, and ELISA were conducted on the groups, which were collected after 24 h, and 48 h of drug exposure. All the experiment was performed at passage 3.

Live/dead assay of hMSCs treated with Dex

20,000 cells per well of 24 well plate was plated with tissue specific hMSCs in serum-free culture medium as described previously for 24 h and 48 h with different concentrations of Dex (1000 ng/ml, 2000 ng/ml, 3000 ng/ml). The Live/Dead assay was performed with Calcein- ethidium homodimer dye (Invitrogen, USA). The sample size for each group’s live/dead assay was kept three (n = 3).

Measurement of metabolic activity by MTT assay of hMSCs treated with dex

The proliferation rate of hMSCs (n = 3) treated without Dex treatment and with Dex treatment was performed. Briefly, 5000 cells per well were seeded in triplicates, and at each termination day (1, 3, 5 and 7) medium was removed and replenish with fresh 180 μl of complete medium and 20 μl of 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, USA) reagent was added. The plate was incubated for 3–4 h at 37 °C and 5% CO2. Followed by removal of medium and formazan crystals were dissolved with 200 μl of DMSO. The solution was collected in a fresh plate and absorbance were taken at 570 and 660 nm using ELISA reader (BioTek, Germany). The technique was performed as per the previously established protocol [24].

Scratch assay

To examine whether different concentration and time points of Dex affects the migratory property of tissue specific hMSCs. Tissue specific hMSCs were plated at 50–60% confluency before preconditioning. Culture plates were incubated at 37 °C and 5% CO2 until confluency reaches 80–90%. Cells were pre-treated with 1000 ng/ml, 2000 ng/ml and 3000 ng/ml of Dex for 24 h and 48 h in serum-free medium. Followed by PBS wash, a scratch was created using p200 pipet on cells monolayer. Again, culture plates were washed with PBS once and replaced with the DMEM-LG with 10% FBS and 1% Penicillin (100 U/ml) + Streptomycin (100 µg/ml). At 0 h, 12 h, 24 h, the scratch area was imaged using phase-contrast microscopy (Olympus, Central Vally, PA) with a 4X magnification lens. The analysis of open area was performed using Image J software. The experiment was performed thrice for all the tissue-specific hMSCs.

Trilineage differentiation

Tissue specific hMSCs were characterized by differentiating cells into osteogenic, chondrogenic and Adipogenic lineages according to the induction protocols described in our earlier published research article [23, 24].

Immunophenotyping

At passage 3, hMSCs were characterized using monoclonal antibodies specific for CD105-APC, CD73-PE, CD29-FITC, CD90-PerCp-Cy5.5, HLA-ABC-APC, HLA-DR-FITC, CD34/45-PE/FITC (BD Pharmingen, France). 50,000 cells were incubated with the respective primary mAb or isotype‐matched control antibody for 40 min in dark. Cells were washed with 1X PBS and analyzed by flow cytometry (BD-LSR-II, San Jose, CA). For HLA-G Flow cytometry studies, the mouse anti-HLA-G1/HLA-G5 MEMG/9 PE antibody (Exbio, Praha, Czech Republic) was used at 1:200 final concentration for 75,000 cells and incubated for 40 min in the dark. For analysis, isotype controls were included. The average of HLA-G was calculated value from 5 donors for all the tissue-specific hMSCs. Acquisition and data analysis were performed using flow cytometry (BD Bioscience) and FACS Diva Software Version 6.2.

Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)

For the isolation of total RNA from cultured tissue-specific hMSCs, the respective T-25 flasks were washed using PBS for the removal of any existing debris or serum. The cells were then trypsinized using Trypsin–EDTA, mixed with complete media, and pellet down at 300 g (Beckman Coulter, California, USA) for 5 min. The cells were transferred into microcentrifuge tubes (MCT) and were then lysed using TRI reagent (Molecular Research Centre, Ohio, USA), 1 ml/ 1 × 106 cells. The total RNA was prepared according to the phenol–chloroform extraction method. The concentration and optical density (OD) of samples were recorded using a Nanophotometer (Implen, Germany). Reverse Transcriptase PCR: cDNA was prepared, using 2 µg/µl of the RNA samples from tissue-specific hMSCs and JEG-3, HeLa cell lines by Reverse transcriptase (RT) enzyme (Promega, USA). Optimization of cDNA using GAPDH: Glyceraldehyde—3 phosphate dehydrogenase (GAPDH) was used in the PCR setup as the housekeeping gene, for the optimization of the prepared cDNA samples of tissue-specific hMSCs, JEG-3 and, HeLa. qPCR was performed in duplicates using SYBR green Master Mix according to the manufacturer’s instruction (Kappa, USA). We calculated the average fold change for PGE-2, IDO and HLA-G value from 5 donors. However, for stemness markers studies, the average value of 3 donors were considered for tissue-specific hMSCs in duplicate using an equation of the standard curve. List of primers (Table 1).

ELISA for PGE-2 and HLA-G

Tissue specific hMSCs, pre-treated with different concentrations of Dex (1000 ng/ml, 2000 ng/ml, 3000 ng/ml) for 24 h and 48 h, were seeded in 6 well plates at 50,000 cells/well and cultured for respective time and dose concentration in 2 ml medium without FBS. Culture supernatant was then collected and the concentration of PGE-2 determined by ELISA (Cayman, USA), whereas HLA-G has been determined by ELISA kit (Biovender, USA). Each group of different Dex concentrations was performed in duplicates (n = 3) for all tissue specific hMSCs.

IDO activity

The biological activity of IDO was calculated by measuring the level of kynurenine in supernatant collected from different preconditioned tissue-specific hMSCs. Briefly, 100 μl of collected cell culture supernatant were added to the Eppendorf tube and 50 μl of 30% trichloroacetic acid (Sigma, USA) were added, the tube was vortexed and centrifuged at 8000 g for 5 min. Then, 75 μl of the supernatant was transferred with an equal volume of Ehrlich reagent (100 mg p-dimethyl benzaldehyde in 5 ml glacial acetic acid) (Sigma, USA) to a 96-well microtiter plate and recorded the absorbance at 490 nm [26]. Experiment was performed at least in three independent setups.

Immunosuppression activity of tissues specific on peripheral blood mononuclear cells (PBMCs)

The study was approved by the Institutional Ethics Committee (IEC) (Ref No. IECPG-345/07.09.2017, (RT-6/29.11.2017). All the samples were obtained after taking the donor’s informed consent. Human Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (Axis-Shield; Oslo, Norway) density gradient centrifugation from blood donated by healthy volunteers. Phytohemagglutinin A (PHA (Sigma, USA); 35 µg/mL) was used to stimulate the activation of human peripheral blood mononuclear cells (PBMCs) before co-culture. For co-culture experiments, hMSCs were treated with Mitomycin C (Sigma, USA); 15 µg/ml and co-cultured (1 × 104 cells/well) with PHA activated hPBMCs (5 × 104 cells/well) in 1:5 ratio in RPMI-1640 medium (Gibco, USA) containing 10% FBS for 3 days in 96-well plates (Costar, USA). Proliferation of hPBMCs were assessed by MTS assay. The 200 μl cell culture supernatant containing hPBMCs were collected in pre-labelled 0.6 ml Eppendorf tube and 20 μl of MTS reagent (Promega, USA) was added in each tube, followed by incubation for 3 h at 37 °C and 5% CO2. Afterwards, tubes were centrifuged at 300 g for 5 min. 200 μl of supernatant was collected from each tube and transferred into the fresh 96 well plate. The absorbance was taken at 490 nm using ELISA reader (Biotek, Germany). Lastly, % decrease was calculated by calculating difference between positive control (Activated PBMCs) and test group (MLR). Then; divided the decrease by the positive control and multiplied the answer by 100.

Statistical analysis

Statistical analysis was performed using Two way-ANOVA, post-test Tukey and t-test in GraphPad Instant software (GraphPad Software, Inc.).

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