Cd146

The adipose tissue was harvested by means of a standard liposuction procedure, and then it underwent a centrifugation of 300× g for 10 min, in order to remove the oil and serous fractions. Then, hASC was released by type IV collagenase (Sigma-Aldrich) digestion from the surrounding connective tissue scaffold for 30 min at 37 °C. Finally, a double series of washing with PBS and centrifugation was conducted to obtain the hASC pellet. The hASC was then cultured on a 10 cm plate in DMEM medium containing 10% FBS and subcultured every two to three days, and passages five to seven were used in the experiments.
HUVECs and hASCs were identified by surface markers, including human CD29, CD90, CD34 (Abcam, Cambridge, MA, USA), CD31, CD44, CD73, CD105, CD146, HLA-DR (Becton–Dickinson, San Jose, CA, USA), using a BD FACSCanto Flow Cytometer (Becton–Dickinson, San Jose, CA, USA). A replicate unstained sample was used as a negative control. Data were analyzed with the BD FACSDiva Version 6.1.3 and the FlowJo 10.1 software (BD Biosciences, Ashland, OR, USA). The differentiation potential of ASCs to adipogenic, osteogenic, and chondrogenic linage was examined using a differentiation–induction protocol and differentiation assay described previously [29 (link),30 (link)].

Cells from the digested cord tissue and cultured in MSCGM were characterized as UCT-MSC based upon morphology and phenotype determined by flow cytometry as separate biological replicates. Antibodies used for phenotypic characterization included CD31, CD14, CD90, CD44, CD83, CD105, CD45, UEA-1, Stro-1, ICAM-1, CD146, CD11b, CD29, CD80, CD117, CD166, CD34, and CD309 (Becton Dickinson, Franklin Lakes, NJ, United States). UEA-1 was purchased from Sigma-Aldrich (St. Louis, MO, United States) and CD51 was purchased from Immunotec (Beckman-Coulter, Miami, FL, United States).
To obtain cord tissue-derived endothelial progenitor cells (UCT-EPC), cells from the digested cord tissue that had been cultured in EGM-2 were labeled with FITC-conjugated Ulex europaeus agglutinin 1 (UEA-1) (Sigma-Aldrich, St. Louis, MO, United States) and isolated with anti-FITC microbeads (MACS Miltenyi Biotec, San Diego, CA, United States). Cells were then phenotypically defined by flow cytometry as separate biological replicates. Endothelial cell lineage was confirmed by visualizing tube formation with the Cultrex “In Vitro Angiogenesis Assay Tube Formation Kit” (Trevigen, Gaithersburg, MD, United States). UCT-EPC were cultured on gelatin-coated flasks using EGM-2 growth media in a humidified 37°C incubator at 5% CO₂.

DFAT cells and MSCs were detached with TrypLE (Thermo Fisher Scientific) and suspended in PBS supplemented with 0.2% FBS and 5 mM EDTA at a density of 5 × 10⁵ cells/mL. The cells were stained for 30 min with the following antibodies: CD44 (PE-Cy7), CD45 (APC-H7), CD73 (V450), CD90 (PE), CD105 (APC), CD29 (APC), CD31 (PE-Cy7), CD34 (PE), CD146 (FITC), CD271 (PE-Cy7), CD140a (V450), and CD140b (PerCP-Cy5.5) (all from Becton Dickinson [BD], Franklin Lakes, NJ, USA). Cell fluorescence was evaluated using a FACSVerse instrument (BD). The data were analyzed using FlowJo software (Tree Star Software, Ashland, CA, USA).