Anti cd34

WJ-MSCs and HUVECs, respectively, at the eighth and the sixth passage, were treated with 0.05% trypsin–EDTA and collected; 10⁶ cells per sample were incubated with 1 μg of the specific antibody, conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), phycoerythrin-cyanine 5.5 (PE Cy5.5), or Alexa Fluor 488 for 30 min at 4°C in the dark. WJ-MSCs were stained using the following antibodies: anti-CD31, anti-CD73, anti-CD13, anti-CD90, anti-CD117, anti-CD14, anti-CD34, anti-CD105, anti-CD146, anti-CD133, anti-CD144, anti-ESA, anti-HLA-ABC, anti-HLA-DR, anti-CD45 (Becton Dickinson [BD], San Jose, CA), anti-CD29, anti-CD44, and anti-CD166 (Ancell, Bayport, MN). HUVECs were stained with anti-CD146 (BD) and anti-CD144 (Acris Antibodies, San Diego, CA). After incubation, cells were washed and acquired with a flow cytometer (FACS Calibur; BD), collecting 10,000 events per sample. Data were analyzed by the FlowJo software v8.8.6 (TreeStar, Ashland, OR). The mean fluorescence intensity (MFI) ratio values were calculated (i.e., dividing the MFI of positive events by the MFI of negative events).^{22 (link),23 (link)}

Flow cytometric analysis was performed on starting samples and at each passage by using the following Mo-Abs: anti-CD71 (allophycocyanin, APC-conjugated, Becton-Dickinson), anti-CD73 (phycoerytrhin, PE-conjugated, Becton-Dickinson), anti-CD90 (allophycocyanin, APC-conjugated, Becton-Dickinson), anti-CD105 (peridinin chlorophyll protein complex, PerCP-conjugated, Becton-Dickinson), anti-CD166 (PE conjugated, Becton-Dickinson), anti-CD45 (FITC-conjugated, Becton-Dickinson) and anti-CD34 (FITC-conjugated, Becton-Dickinson).
At the end of the culture time, MSC were checked for viability by using (7-amino-actinomycin D) 7-AAD (Becton-Dickinson) and a Mo-Ab identifying CD105, which represents a MSC constitutive antigen. Viable MSC were identified as CD45-, CD105+, 7-AAD- cell events.

The phenotype of differentiating MKs and PLTs was analyzed using FITC or PE conjugated monoclonal antibodies anti-CD34 (Becton Dickinson) and anti-CD61 (BioLegend). Cells were incubated with antibody (diluted 1:100 for 15 min), washed with PBS, and analyzed in a cytofluorimeter (Epics XL–MCL Coulter).

Vertebra samples were collected from mice, fixed and serial 5-μm sections were prepared in paraffin. Immunohistochemistry was performed with primary anti-VEGF antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-α-actin (for TM mice) and anti-CD34 (for transplante mice) (Becton Dickinson, San Jose, CA, USA) at 1:500, 1:800 and 1:50, respectively. Peroxidase activity was revealed by 3,3′-diaminobenzidine as chromogen. Alpha-actin and CD34 expression was used for assessing microvessel density (MVD), quantified as the number of microvessels/mm² after analyzing three high-power fields (400 ×).

Synovial membrane specimens were digested with 0.2% type I collagenase (Thermo Fisher) at 37°C overnight, which were further collected by centrifugation and seeded in a high-glucose DMEM medium (Thermo Fisher, Waltham, MA, United States) supplemented with 10% FBS for 4 days to allow cell attachment. The medium was refreshed every 3 days, and at day 14, SMSCs were obtained. After blocking with human BD Fc Block™, SMSCs were stained with the following antibodies (Becton Dickinson): anti-CD34, anti-CD44, anti-CD45, anti-Sca-1, and anti-CD105 antibodies to confirm the phenotype using Guava^® easyCyte™ flow cytometer (Merck-Millipore, Billerica, MA, United States). At passage 3, SMSCs were switched to osteogenic differentiation medium (Sigma-Aldrich, St. Louis, MO, United States) for 2 weeks or StemPro Adipogenesis Differentiation Kit (Gibco) for 4 weeks. The miR-212-5p mimic or mimic-negative control (NC) (Sigma-Aldrich) were transfected with Lipofectamine^® 3,000 (Thermo Fisher) at the concentration of 100 nM according to the manufacturer’s instructions. The following sequences were used: miR-212-5p mimic (sense, 5′-ACC​UUG​GCU​CUA​GAC​UGC​UUA​CU-3'; and antisense, 5′-UAA​GCA​GUC​UAG​AGC​CAA​GGU​UU-3′), mimic-negative control miRNA (sense, 5′-UUC​UCC​GAA​CGU​GUC​ACG​UTT-3'; and antisense, 5′-ACG​UGA​CAC​GUU​CGG​AGA​ATT-3′).