Characterization of hUCMSCs Differentiation

The hUCMSCs were purchased from Haixing Biosciences (Suzhou, Jiangsu, China). The hUCMSCs were cultured in accordance with a previously described method[45 (link)]. Afterward, the adipogenic, osteogenic, chondrogenic capacity of these cells was performed. Passages 3–7 of hUCMSCs were cultured in osteogenic, adipogenic or chondrogenic differentiation medium (Cyagen, China, HUXUC-9004) as described by manufacturer. Oil red O staining, Alizarin red staining and Alcian blue staining were used to evaluate adipogenesis, osteogenesis, and chondrogenesis. Detection of hUCMSCs surface markers by a FACSVerse instrument (BD Bioscience, San Jose, CA, USA) is performed as follows. The hUCMSCs were stained with human anti-CD14, anti-CD19, anti-CD34, anti-CD45, anti-CD73, anti-CD90, anti-CD105, or anti-HLA-DR. Identical concentrations of PE-conjugated mouse IgG isotype antibodies were used as negative controls (all from BD Biosciences). Data were analyzed using the software FlowJo V10 (FlowJo).