Anti cd105

The surface antigen expression of MSCs was determined by flow cytometry. The following antibodies were used: anti-CD29 (Biolegend, 303003), anti-CD45 (Biolegend, 304011), anti-CD73 (Biolegend, 344003), anti-CD90 (Biolegend, 328107) and anti-CD105 (Biolegend, 323205). Aged MSCs were transfected with either a lentivirus containing MIF (MIF-aged MSCs) or a control vector (aged MSCs), as previously described [48 (link)]. The transfection efficiency was examined by fluorescence microscopy and Western blotting. The capacity of MSCs to differentiate into osteocytes and adipocytes was examined as previously reported [48 (link)].

BMSCs cells were isolated from Wistar rats (weighted 80–100 g; specific pathogen-free grade; Vital River Laboratories, Beijing, China) and cultured as previously described (Hussain et al., 2016 (link)). Rats were anesthetized by intraperitoneal (IP) injection of chloral hydrate (350 mg/kg), and the femur and tibia of rats were excised and bone marrow cells were slowly flushed out from the marrow cavity using DMEM medium (Gibco, Grand Island, NY, USA). Cell was cultured with DMEM supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA). Non-adherent cells were removed after 48 h. Approximately 14 days later, the adherent stromal cells become confluent and were designated passage 0 (P0). The BMSCs were expanded and passaged to P3, at which point they were harvested and employed in the present study. Animal experiments performed in this study were all approved by the Animal Ethics Committee of Southern Medical University. Precautions were taken to minimize suffering and the number of animals used in each experiment.
BMSCs were identified by the expression of specific membrane markers using cytofluorimetric analysis with a flow cytometer (Beckman Coulter, CA, USA; Yu et al., 2013 (link)). Cells were incubated for 20 min at 4°C with the following antibodies: anti-CD29, anti-CD90, anti-CD105 and anti-CD45 (Biolegend, CA, USA).

Flow cytometric analyses were performed with Gallios (Beckman Coulter) flow cytometers, and the data were analyzed with the FlowJo (Treestar) software packages.
MSCs were incubated with anti-CD90, anti-CD105, anti-CD73, anti-CD44, anti-CD11B, anti-CD34, anti-CD19, anti-CD45 and anti-HLA-DR antibodies to examine MSC surface markers, which were purchased from BioLegend. Anti-CD3, anti-CD4, anti-CD8, anti-TNF-α and anti-IFN-γ antibodies were used to examine stain T cells. Propidiumiodide (PI; BD) and Annexin V (BD) were used to stain apoptosis cells. Anti-CD86, anti-F4/80 and anti-CD45 antibodies were used to stain macrophages.

Isolation and identification of rat TSCs were performed as previously described [16 (link)]. Briefly, 0.3% pentobarbital sodium (Sigma, 30 mg/kg) was used for intraperitoneal anesthesia in rats. In sterile conditions, the tendon tissues were then removed, carefully dissected, cut into pieces, and digested in 3 mg/mL of type I collagenase (Sigma-Aldrich, St. Louis, MO, USA). After a 70-μm cell filter filtration, the suspension turned into a single cell suspension, which was then cultured in Dulbecco’s modified Eagle’s medium (Gibco, Invitrogen, NY, Invitrogen Corporation, Grand Island, USA) containing 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel) and 1% penicillin-streptomycin antibiotic mixture (Beyotime, Shanghai, China). Cells were subcultured at 80% confluence. Cells at passages three were incubated with fluorescein isothiocyanate-conjugated antibodies (anti-CD90, anti-CD105, anti-CD44, anti-CD11b, and anti-CD106) (Biolegend San Diego, CA, USA) through flow cytometry. The multilineage differentiation potential of TSCs was determined by inducing the differentiation of cells in passage 3 into osteocytes, adipocytes, and chondrocytes (all the differentiation media were from Cyagen).

ASCs were isolated from adipose tissue from healthy donors and AAA patients as previously reported [15 (link)]. Briefly, adipose tissue (1-5 g) was cut into small pieces and digested with enzyme and subsequently plated on 10 cm culture dishes. After 48 hours, nonadherent cells were washed off and the remaining cells cultured with DMEM/low glucose (Gibco) medium supplemented with 10% FBS (Life Technologies, 16000), 0.1 mM 2-mercaptoethanol (Life Technologies, 21985023), NEAA (Life Technologies, 11140050), and 0.1% Penicillin/Streptomycin (Life Technologies, 15140122). The ASCs at passages 3~4 were used in the current study. Both H-ASCs and AAA-ASCs were passaged at 3-day intervals and the same cell number (100,000 cells per 6 cm dish) plated. Population doubling was evaluated at each passage.
Surface markers of H-ASCs and AAA-ASCs were determined using flow cytometry. Antibodies including anti-CD31 (BioLegend, 303111), anti-CD45 (BioLegend, 304011), anti-CD73 (BioLegend, 344003), anti-CD90 (BioLegend, 328107), and anti-CD105 (BioLegend, 323205) were used. The differentiation capacity of H-ASCs and AAA-ASCs into adipocytes and osteocytes was examined as previously described [16 (link)].