Cd90 apc

In this work, we used rat and human bone marrow-derived mesenchymal stem cells (MSCs). Human MSCs 4–6 passage were taken from the culture bank of Research Institute of Clinical and Experimental Lymphology—Branch of the ICG SB RAS (RICEL–branch ICG SB RAS). Rat MSCs were harvested from the femoral and tibia bones of Wistar rats (8 weeks old) according to standard protocol. Cells in the fourth passage were used for phenotype screening of MSCs by flow cytometry (CytoFLEX S, Beckman Coulter, Brea, CA, USA) using antibodies to CD90FITS, CD90APC, CD29APC, CD73FITS (BD Biosciences, San Jose, CA, USA), and hematopoietic cell marker CD45FITS (BD Biosciences, San Jose, CA, USA).
All animal procedures were conducted in accordance with ethical standards of the international, institutional animal care guidelines. The Committee of Ethics in Animal Research of the Federal Research Center of Fundamental and Translational Medicine reviewed and approved all animal procedures (No. 10 of 26/03/2019).

The adipose tissue stem cell line RESSTORE01 (Master Cell Bank/Stock n°1—Donor RESSSTORE01, Batch n°: 591133643763) was cultured in the growth medium Alpha MEM (Gibco, Life technologies) supplemented with 5% human platelet lysate (Stemulate, Cook Medical, USA) and 1% Penicillin-Streptomycin (Lonza, Belgium). The medium was changed twice each week and cells were passaged when they reached 100% confluence. The cells were detached with TrypLE Select (Life Technologies™, Thermo Fisher Scientific) for 10 min at 37°C and then centrifuged at 1,000 rpm for 5 min. The RESSTORE01 cell phenotype was analyzed with flow cytometry (FACSAria Fusion Cell Sorter, BD Biosciences) at passage PX +1. Monoclonal antibodies against CD19-phycoerythrincyanine (PE-Cy7), CD45RO-allophycocyanin (APC), CD73-PE, CD90-APC (BD Biosciences), CD11a-APC, CD105-PE (R&D Systems Inc., Minneapolis, MN, USA), CD34-APC and HLA-DR-PE (Immunotools GmbH, Friesoythe, Germany) were used. The cells expressed (>95%) surface markers CD73, CD90, and CD105 and lacked the expression (<2%) of CD11a, CD19, CD34, CD45, and HLA-DR. Cells at passages PX +2/3 were used for the study.

At the day of infusion, ASCs monolayer were dissociated as described above and 1 × 10⁶ cells/kg of the donor patient were suspended in 5 ml of saline solution with 50% albumin and 5% ACD (Anticoagulant Citrate Dextrose Solution). Cell suspension was sent to the hospital in cooler with recycled ice.
An aliquot of cells was evaluated after transportation for monitoring viability and phenotype of ASCs by flow cytometry. Cells were washed with PBS-BSA 3% (bovine serum albumin) and incubated at 4°C for 30 min with the following monoclonal antibodies conjugated to fluorescent dyes: CD105-FITC (fluorescein isothiocyanate), CD73-PE (phycoerythrin) and CD90-APC (allophycocyanin), all from BD Biosciences, Franklin Lakes, NJ, USA. Unstained cells were used as controls. Then, cells were washed with PBS-BSA 3% and incubated with 7AAD (7 Amino Actinomycin D). Twenty thousand events were acquired in FACSAria III cytometer (BD Biosciences) and data were analyzed using FACSDiva 8.0 software (BD Biosciences). The percentage of viable cells was estimated by 7AAD exclusion.
Patients that received ASCs were admitted into hospital in the day of the infusion. A single dose of ASCs was infused in a peripheral upper arm vein during 15–20 min. Patients were discharged from hospital 24 h after infusion. Patients started taking oral cholecalciferol 2,000 IU in the same day.

The MSC-like cells were harvested by trypsinization (Gibco) and incubated with 1% bovine serum albumin (BSA) for 1 h at 4 °C to block nonspecific Fc-mediated interactions, then incubated in the dark at 4 °C for 30 min with 400 μl of CD45-FITC (BD Biosciences, 340664, Franklin Lakes, NJ), CD34-APC (BD, 555824), CD105-FITC (BD, 561443), CD73-PE (BD, 561258), and CD90-APC (BD, 559869) antibodies. The cells were stained with PE- or FITC-labeled IgG as an isotype control. The cells were evaluated by a FACSCalibur flow cytometer (Becton–Dickinson, San Jose, CA) and analyzed with FlowJo software (Tree Star Inc., Ashland, Oregon, USA). The percentage of stained cells was calculated relative to the isotype control.

MSCs were analyzed using the following antibodies: CD73-PE, CD105-PE, CD14-PE, CD45-FITC, CD90-APC (BD Biosciences), CD106-Pe-Cy5, CD34-Pe-Cy5 (BD Biosciences), HLA-DR-Qdot-605 (Invitrogen), mouse mAb specific to FAP (eBioscience) and PE-conjugated anti-mouse (BD Biosciences). Isotype-matched, fluorochrome-conjugated, mAb were used as negative controls.
Cultured SCS were stained, data acquired and analyzed as previously described using the following antibodies [11] (link); Surface: CD3-Qdot-605, CD4-Qdot-655 (Invitrogen), CXCR5-Alexa-488, CD25-APC-Cy7, PD-1-Brilliant Violet-421; Intracellular: Bcl-6-PE-CF594, active caspase-3-PE (BD Biosciences), and FoxP3-PE-Cy7 (eBioscience).
FL T-cell subsets were flow cytometrically sorted using the following markers; T-cell: DAPI⁻CD3⁺CD4⁺CD19⁻; TFH: Propidium iodide⁻CD3⁺CD4⁺CXCR5⁺PD-1⁺CD25⁻. Following the sort, an aliquot of TFH were permeabilized and stained with Bcl-6 and FoxP3 to confirm the TFH phenotype. TFH cells were defined as CD3⁺CD4⁺CXCR5⁺PD-1⁺CD25⁻Bcl-6⁺; TFR were defined as CD3⁺CD4⁺CXCR5⁺PD-1⁺CD25⁺Bcl-6⁺FoxP3⁺ and Tregs were defined as CD3⁺CD4⁺CD25⁺FoxP3⁺.

The phenotype of ASCs was assessed with flow cytometry (BD FACSAria™ Fusion Cell Sorter; BD Biosciences) at passage 5. Monoclonal antibodies against CD markers (see Additional file 1: Table 2) CD19-phycoerythrincyanine (PE-Cy7), CD14- PE-CF594, CD34-PE-CF594, CD45RO- allophycocyanin (APC), CD54-brilliant violet 711 (BV711), CD73-PE-Cy7, CD90-APC, CD146-PE, HLA-DR-BV421 (BD Biosciences), CD105-fluorescein isothiocyanate (FITC), and HLA-ABC-PE (ImmunoTools GmbH, Friesoythe, Germany) were used. Multicolor staining was used to assess the immunophenotype of ASCs. Ten thousand cells were analyzed, and unstained cells were used to adjust the cytometer. Compensation for all the antibodies was performed using Compensation Plus beads (BD Biosciences).