Hla abc

For Western blotting, PIK3CA (catalog 4249S), pAKT T308 (catalog 9275S), pAKT S473(catalog 4051S), and β-actin (catalog 4970S) were purchased from Cell Signaling Technology. Also used were AKT (Santa Cruz, sc-8312), β-tubulin (Sigma-Aldrich, T4026), and EGFR (Epitomics, 1902-1). Anti-mouse IgG secondary antibody HRP (catalog 62-6520) and anti-rabbit IgG secondary antibody HRP (catalog 31460) were from Thermo Fisher Scientific. For flow cytometry, we used anti-mouse FcR block (catalog 101302), CD90.2 (clone 30-H12), anti-mouse CD80 (clone 16-10A1), H-2K^b (clone AF6-88.5), Human Trustain FcX (catalog 422301), and anti-human CD80 (clone 2D10) from BioLegend, and HLA-ABC (eBioscience, clone W6/32) and annexin V (Invitrogen, catalog A23204). For histology analyses, we used F4/80 (catalog ab111101), CD3 (catalog ab16669), and CD4 (catalog ab183685) from Abcam, and CD8 (catalog 98941) from Cell Signaling Technology.

This study was approved by the Navy General Hospital Ethical Committee. With the consent of the parents, human umbilical cord Wharton’s jelly was collected from infants delivered by full-term normal labor. The cells from the Wharton's jelly tissue were isolated as described previously [26 (link)]. Briefly, after removal of blood vessels and epithelium, the Wharton’s jelly was cut into pieces about 1.5–2.5 mm³, and digested for 18 hours at 37 °C with 0.2 mg/ml collagenase (Sigma St. Louis, Missouri, USA) solution in serum-free medium containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin B. The isolated cells were suspended in Dulbecco's modified Eagle’s medium (DMEM)/F12 nutrient mixture containing 20 % fetal bovine serum (FBS). When WJCs were cultured to reach 80–90 % confluence, they were passaged and seeded at a density of 4 × 10³/cm² in DMEM/F12 with 10 % FBS. WJCs at passage 3 were taken for flow cytometry analysis as described previously [42 (link)]. The antibodies used were phycoerythrin (PE)-conjugated CD105, CD73, CD45, CD29, CD166, human leukocyte antigen (HLA)-DR, HLA-ABC, and fluorescein isothiocyanate (FITC)-conjugated CD34 and CD90 (all eBioscience, Santiago, California, USA).

Fluorochrome (FITC or PE or APC)‐conjugated anti‐human antibodies CD73, CD90, CD105, CD71, CD29, CD31, CD34, CD45, HLA‐ABC, and HLA‐DR were purchased from eBioscience (CA, USA). AT‐MSCs were harvested, washed with PBS, and incubated with antigen‐specific antibodies for 30 min at room temperature. The non‐specific staining was controlled by isotype‐matched antibodies. Flow cytometry was performed on a FACSCanto II (BD Biosciences, CA, USA) with FACSDiva software for data acquisition (BD Biosciences). Data were analyzed with Summit software v4.3 (Beckman Coulter, CA, USA).

Human MAPCs (n = 2) used in this study were isolated by ReGenesys BVBA (Athersys affiliate; Heverlee, Belgium) from the bone marrow of a 30-year-old female and a 45-year-old male volunteer, with informed consent and ethical approval. Isolation and culture of the cells was carried out as outlined in electronic supplementary material (ESM) Methods [22 (link)].
Phenotypic analysis of the human MAPCs was performed using fluorochrome-conjugated antibodies recognising cluster of differentiation (CD) 3, CD31, CD34, CD40, CD44, CD86, CD105, fetal liver kinase 1 (Flk1), HLA-ABC and HLA-DR (ebioscience, San Diego, CA, USA). Acquisition was done by using a Gallios multicolour flow cytometer (Beckman Coulter, Suarlée, Belgium). For analysis of the samples, FlowJo version 10.1 (Tree Star, Ashland, OR, USA) software was used.
Cell-free supernatant fractions were assayed for a pro-inflammatory, cytokine, chemokine, angiogenesis and vascular inflammation panel (see ESM Methods) by multiplex electrochemiluminescence (Meso Scale Discovery, Rockville, MD, USA) as per manufacturer’s protocol.
The angiogenic potential of human MAPCs was examined in the chick chorioallantoic membrane (CAM) as described [23 (link)].

P3 MenSCs-DGC (n=5) and MenSCs-RLB (n=5) were used for the immunophenotyping analysis. Mouse anti-human monoclonal antibodies: FITC-conjugated CD29 (Cat#: 11-0299-42, dilution ratio 1:20, eBioscience), CD44 (Cat#: 11-0441-82, dilution ratio 1:20, eBioscience), CD73 (Cat#: 11-0739-42, dilution ratio 1:20, eBioscience), CD90 (Cat#: 11-0909-42, dilution ratio 1:20, eBioscience), HLA-ABC (Cat#: 11-9983-42, dilution ratio 1:20, eBioscience), HLA-DR (Cat#: 11-9956-42, dilution ratio 1:20, eBioscience), CD34 (Cat#: 11-0349-42, dilution ratio 1:20, eBioscience), CD45 (Cat#: 11-9459-42, dilution ratio 1:20, eBioscience) and PE-conjugated CD105 (Cat#: 12-1057-42, dilution ratio 1:20, eBioscience) were used. As negative controls, isotype PE (Cat#: 12-4714-42, dilution ratio 1:20, eBioscience) and FITC-conjugated IgG (Cat#: 31505, dilution ratio 1:20, eBioscience) were used. The cell suspensions (1×10⁶ cells) were washed twice with PBS and incubated with the monoclonal antibodies at 4°C in the dark for 30 min. After washing with PBS, the samples were resuspended and analyzed using a Cytomics FC 500 MPL cytometer (Beckman Coulter).

ADSC were grown until confluent, trypsinized and pelleted by centrifugation at 200 g for five minutes. For fluorescence-activated cell sorting (FACS) analysis, approximately 2.5 × 10⁵ cells were resuspended in 100 μl FACS buffer containing 10% FBS in PBS. For FACS analysis of surface markers, each sample was incubated for 30 minutes at 4°C with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies against the following surface markers: CD45, CD34, CD11b, CD31, CD19, CD90, CD44, CD71, CD29, CD73, STRO-1, HLA-ABC, HLA-DR, CD117, CD105, CD106 (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. After incubation, the labeled cells were diluted with 2 ml of FACS buffer, pelleted and resuspended in 500 μl of FACS buffer. Generally, approximately 10⁴ cells were analyzed per sample using the BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Results were analyzed using FlowJo software.