Bm0135

All experimental protocols were reviewed and approved by the Experiment and Ethics Committees of Shanghai Stomatological Hospital, Fudan University (20170007). PDLSCs were isolated from healthy premolars extracted from two donors (aged 11 and 13 years) for orthodontic reasons. Briefly, the periodontal ligament tissues were minced and seeded in growth medium consisting of α-minimum essential medium (GIBCO, Carlsbad, CA, USA), 10% fetal bovine serum (GIBCO), 1% l-glutamine (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic-antimycotic solution (GIBCO) at 37°C in a 5% CO 2 atmosphere. A single-cell suspension of the primary cultures was diluted to 10 cells/ mL and seeded into 96-well plates (100 µL/well). Using this limiting dilution technique, all colonies were collected and expanded as PDLSCs. 19 To evaluate the colony-forming efficiency, a single-cell suspension (2 × 10 2 cells) was seeded into a 6-cm diameter culture dish. The cells were stained with 0.1% crystal violet (Beyotime, Jiangsu, China) after 10 days of culture. 19, 20 The isolated PDLSCs were characterized using immunohistochemical staining for expression of vimentin (BM0135, Boster, Wuhan, China), immunofluorescence staining for expression of STRO-1 (ab102960, Abcam, Cambridge, UK) and assessment of induced osteogenic differentiation. PDLSCs at passages 4-5 were used in this study.