Cd105 fitc

Cells were cultured in 6-well plates as described above. After 72 h culture, cells with approximately 80% confluence were collected with Accutase (BDSciences, San Diego, CA, USA) and analyzed for CD105 and CD133 marker expressions. Cells were stained with (i) CD105-PE (BD Pharmingen, San Diego, CA, USA, clone 266) plus CD133/1-APC (Miltenyi Biotec, Bergisch Gladbach, Germany, clone AC133); or (ii) CD105-FITC (Biolegend, San Diego, CA, USA, clone 43A3) plus CD133/2-APC (Miltenyi Biotec, Bergisch Gladbach, Germany, clone 293C3) according to the manufacturers’ protocols with the appropriate isotype controls. Subsequently, the samples were acquired in a FACSCalibur system. For each measurement, at least 10,000 cells were acquired. Data were analyzed by Flowing Software (Turku Centre for Biotechnology, Turku, Finland).

Cells were detached with Accutase (Sigma-Aldrich; 5 min at 37 °C) and then incubated with the following mouse anti-human mAbs for 30 min on ice: CD73-Pacific Blue™, CD90-FITC, CD105-FITC, CD166-PE, CD34-APC, CD45-PerCP, CD117-PE (all from BioLegend) and CD133-APC (Miltenyi Biotec). After two washing steps (PBS with 2 % FBS), the sample tubes were acquired on a BD FACSAria III (BD Biosciences) and at least 10.000 events were recorded. Data were analyzed with BD FACSDiva and FlowJo V10 software.

Cells were labeled for 40 minutes at 4°C with the following antibodies: CD146-APC (1 : 100, R&D Systems, Minneapolis, MN, USA); NG2-PE (1 : 100, R&D Systems); PDGFRβ-PE (1 : 100, R&D Systems); CD31-FITC (1 : 100, BioLegend, San Diego, CA, USA); CD45-APC (1 : 100, BioLegend); CD105-FITC (1 : 100, BioLegend); CD90-PE (1 : 100, BD Biosciences, San Jose, CA, USA); and CD73-PE (1 : 100, BioLegend). Analyses were performed using Guava EasyCyte 5HT™ Flow Cytometer and GuavaSoft 2.1 software (Millipore, Billerica, MA, USA).

Approximately 10⁵ MSCs were incubated with 10 μl monoclonal antibody conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): CD105-FITC (Biolegend, ImTec, Antwerpen, Belgium), CD90-PE (Biolegend), CD73-PE (Biolegend) and CD166-PE (Biolegend). Unbound antibody was washed after 15 min with 3 ml PBS (Gibco). The cell pellet was resuspended in 0.5 ml PBS (Gibco). Samples were analyzed with the flow cytometer Macs Quant (Miltenyi Biotec, Bergisch Gladbach, Germany) with 10,000 events recorded for each condition. The morphology of cultured cells was evaluated using light microscopy.

Cell surface antigens on cells were evaluated with FACS. The cells were dissociated with 0.05% trypsin/EDTA (Highclone), washed with PBS, fixed with 4% paraformaldehyde, permeabilized with Triton X‐100, and blocked with a BSA mixture. The samples were then stained with antibodies against human octamer‐binding transcription factor 4 (OCT4; R&D systems, 1:200), stage specific embryonic antigen 1 (SSEA1; R&D systems, 1:200), cluster of differentiation 31 (CD31‐FITC; Miltenyi Biotec, 1:100), CD34 (CD34‐PerCP, BioLegend, 1:100), human leukocyte antigen ‐ DR isotype (HLA‐DR, HLA‐DR‐APC, BioLegend, 1:100), CD73 (CD73‐PE, BioLegend, 1:100), CD90 (CD90‐APC, BioLegend, 1:200), CD105 (CD105‐FITC, BioLegend, 1:150), integrin α5 (Santa Cruz, 1:200), integrin α11 (Abcam, 1:200), integrin β1(Abcam, 1:200), and integrin β5 (BioLegend, 1:200) for 30 min or 1 h at 4 °C. Samples were subsequently stained with fluorescently labeled secondary antibodies (Alexa Fluor‐488 or 594 conjugated goat antirabbit (Abcam, 1:400), Alexa Fluor‐488 or 594 conjugated goat antimouse (Abcam, 1:400)) for 30 min at 4 °C. The corresponding mouse/rabbit isotype antibodies (Abcam, 1:200) were used as controls. Cell immunotypes were determined with the Accuri C6 flow cytometer (BD Biosciences) and the percentage of expressed cell surface antigens was calculated for 10 000 gated‐cell events.

Ex vivo expanded BM-MSCs were characterized for expressing MSC official markers by flow cytometry and their ability to differentiate into mesodermal cell types, as indicated by the International Society for Cellular Therapy. In detail, BM-MSCs were trypsinized, washed in phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS), and stained with the following antibodies for 15 min at room temperature: CD105 FITC, CD73 PE, CD90 PE, CD146 PB, CD45 FITC, CD31 FITC (BioLegend, San Diego, CA, USA). After washing twice in PBS + 2% FBS, cells were analyzed using the BD FACSCanto^TM (BD Life Sciences, San Jose, CA, USA). Data were analyzed using FlowJo™ v10.8 Software (BD Life Sciences, Ashland, OR, USA) and represented as a percentage of positive cells on total cells.
BM-MSCs were induced to differentiate into osteoblasts and adipocytes using the proper differentiation medium for human cells (Miltenyi Biotec, Bergisch-Gladbach, Germany). After 14 days, adipogenic and osteogenic differentiation was evaluated by Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) and Alizarin Red staining (Sigma-Aldrich, St. Louis, MO, USA), respectively.

Samples from four women were used in this step. The cells were evaluated according to the criteria for characterization of MSCs^{33 (link)}. Regarding adhesion to polystyrene and formation of fibroblast-like forming colonies, immunophenotypic¹⁵ characterization was done by flow cytometry by labeling the cells with the following antibodies: CD73-PECy7, CD90-FITC, CD105- FITC, CD19-PE Cy7, CD34-APC Cy7, CD45-APC, HLA-DR-PERCP Cy5.5, CD44-PERCP Cy5.5, CD49c-PE, CD151-APC, CD166-PE, CD117–APC, SSEA4-FITC, Oct4-PE (Biolegend, San Diego, CA), and NANOG-PERCP Cy5.5 (BD Pharmingen). The analysis was done in BD FACSCanto or BD FACSVerse and the results were analyzed using BD FACSDiva.
Analysis of differentiation into the three main mesenchymal lines was performed using adipogenesis, chondrogenesis, and osteogenesis StemPro differentiation kits (Gibco), according to the manufacturer's protocol. 0.5% oil Red O (Sigma-Aldrich), Alcian blue (Sigma-Aldrich), and Alizarin red (Sigma-Aldrich) were used respectively to observe adipogenesis, chondrogenesis, and osteogenesis.