Anti human cd73

FITC or phycoerythrin (PE)-conjugated monoclonal antibodies anti-human CD73 and CD90 were from BD Biosciences (Franklin Lakes, NJ, U.S.A.). Monoclonal antibodies anti-mouse CD34 were from Biolegend (San Diego, CA, U.S.A.). Polyclonal antibody anti-human Lgr6 was from Novus Biologicals (Littleton, CO, U.S.A.). Polyclonal antibodies anti-human KGF and bFGF) were from R&D Systems (Minneapolis, MN, U.S.A.). Atelocollagen membranes (CM) were purchased from Cosmo Bio (Tokyo, Japan). α-MEM medium was from Life Technologies (U.S.A.) and Chang medium was from Irvine Scientific (U.S.A.).

For immunophenotype studies, dual-color immunofluorescence was performed using the following panel of phycoerythrin (PE)-conjugated or fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies: anti-human CD14, anti-human CD13, anti-human HLA-DR, anti-human CD34, and anti-human CD73 (BD Biosciences, Buccinasco, Italy); and anti-human CD105, anti-human CD44, anti-human CD45, anti-human CD90, and anti-human CD29 (Chemicon, Temecula, CA, USA). Negative controls were isotype-matched irrelevant monoclonal antibodies (BD Biosciences). MSCs (5 × 10⁵) were incubated in the dark for 15 minutes at 4 °C in PBS–1 % bovine serum albumin (BSA). Cells were rinsed in PBS and analyzed using a BD Accuri C6 flow cytometer (BD Biosciences). A minimum of 10,000 events was collected in list mode on FCS Express 4 Flow Research Edition Software (De Novo software, Glendale, CA, USA).

hAMSC and hAEC specific surface antigens were stained with PE-conjugated antibodies of anti-human-CD-105, anti-human-CD-29, anti-human-CD-44, anti-human-CD-73, anti-human-CD-90, anti-human-CD-34, anti-human-CD-45, anti-human-EpCam, or anti-human-CD-166 (BD, USA) or their corresponding isotype control. The stained cells were then analyzed respectively by fluorescence-activated cell sorting (FACS). The detailed instruction is the same as the FACS analysis already described. Differentiation ability of hAMSCs was tested using hAMSC differentiation kits for culture (Thermo, USA).