Pe conjugated mouse anti human cd105

hPDLSCs were stained with one of the following monoclonal antibodies (all from eBiosciences, San Diego, CA, USA): phycoerythrin (PE)-conjugated mouse anti-human CD29, PE-conjugated mouse anti-human CD90, PE-conjugated mouse anti-human CD105, PE-conjugated mouse anti-human CD146, PE-conjugated mouse IgG1 K isotype control, fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD14, FITC-conjugated mouse anti-human CD31, FITC-conjugated mouse anti-human CD34, FITC-conjugated mouse anti-human CD45, FITC-conjugated mouse IgG1 K isotype control.

hPDLSCs and hGMSCs were characterized by analyzing the expression of characteristic cell surface markers. Single cell suspensions were stained 1 : 10 with one of the following antibodies (all from eBioscience, San Diego, USA) for 20 min: phycoerythrin- (PE-) conjugated mouse anti-human CD90, PE-conjugated mouse anti-human CD105, PE-conjugated mouse anti-human CD146, PE-conjugated mouse anti-human CD29, PE-conjugated mouse anti-human CD73, fluorescein isothiocyanate- (FITC-) conjugated mouse anti-human CD34, FITC-conjugated mouse anti-human CD45, and FITC-conjugated mouse anti-human CD31. After resuspending cells in 200 μl of FACS buffer (3% BSA, 0.09% sodium azide, in 1x PBS), an argon laser was used, exciting the fluorescence at 488 nm. The percentage of positive cells for each investigated surface marker was determined using the FACScan Flow Cytometer (Becton Dickinson, Franklin Lakes, USA).

To confirm whether HA preconditioned iMSCs express the typical cell surface markers for MSCs, HA-iMSCs were trypsinized and washed twice prior to resuspension in PBS containing 2% FBS. Cells were adjusted to 1 × 10⁶ in 100 µL of cell suspension. For cell surface labeling, cell suspensions were incubated at 4 °C for 30 min with antibodies (dilution, 1:20). APC conjugated mouse anti-human CD73, PE-conjugated mouse anti-human CD105, FITC-conjugated mouse anti-human CD45, PE-conjugated mouse anti-human CD31, and APC conjugated mouse anti-human CD34 antibodies were supplied by eBioscience (Waltham, MA, USA) and, APC-Cy7-conjugated mouse anti-human CD90 was supplied by BioLegend (San Diego, CA, USA). To characterize surface proteins of EVs, HA-iMSC-EVs analysis was performed using human MACSPlex Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Flow cytometric analysis was conducted using an Attune NxT flow cytometer (Thermo Fisher Scientific).