Isolation and Characterization of ADSCs

Human adipose tissues and foreskins were obtained in accordance with procedures approved by the Ethics Committee at the Tongji Medical Collage of Huazhong University of Science and Technology. Primary fibroblasts were isolated from foreskins derived from routine pediatric circumcisions, using previously described protocols [26 (link)]. ADSCs were isolated and cultured in accordance with our established protocol [27 (link)]; ADSCs at passages 3–8 were used for experiments. Flow cytometry was conducted to identify ADSC phenotypes. ADSCs were incubated for 1 h with anti-CD34-BV421, anti-CD44-APC, anti-CD105-PE, anti-CD73-FITC, anti-CD31-FITC, and anti-CD90-FITC antibodies (all from Biolegend, San Diego, CA, USA); fluorescence was observed thereafter. For multi-lineage differentiation potential assays, ADSCs were incubated separately with adipogenic medium and osteogenic differentiation medium (Cyagen Biosciences Inc., Guangzhou, China). Cells were stained with Oil Red O and Alizarin Red, respectively. Subsequently, the stained cells were imaged using a microscope.