70 mm strainer

Colonies were dissociated to single cells by treatment with Accutase (Fisher) for 3–5 minutes. Cells were washed with 10 ml mTeSR (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) media, filtered on a 70 mm strainer (BD Bioscience), spun down for 5 minutes at 1,000 rpm, and resuspended in FACS buffer (1× PBS, 0.1% bovine serum albumin, EDTA) to a concentration of 1 × 106 cells per 100 µl. Cells were blocked with 0.8% mouse IgG (Invitrogen) then incubated with TRA‐1–60 (BD Biosciences) and SSEA4 (BD Biosciences) or TRA‐1–60 live stain (Stemgent) antibodies for 30 minutes on ice and protected from light. The cells were then washed with fluorescence‐activated cell sorting (FACS) buffer and resuspended in FACS buffer + DAPI. Flow‐cytometry analysis was performed on a BD FACS AriaII cell sorter. Compensation beads (BD Biosciences) were used to ensure proper staining patterns during data acquisition. Dead cells and doublets were gated out, and PE‐TRA‐1–60 versus FITC‐SSEA4 was then used to identify a double positive and double negative (fibroblast) population, which was then purity sorted before cells were single cell sorted into individual wells of 12‐8 strip PCR tubes with 5 µl of CellsDirect 2× reaction mix solution (Invitrogen, Carlsbad, CA, http://www.invitrogen.com).

This study was approved by the Research Ethics Committee of School of Stomatology, Shandong University. Extracted healthy first or second premolars and third molars from donors aged from 18 to 23 years were collected with their and their parents' informed consent. The associated information was shown in Table S1. All donors were healthy and had no systemic diseases. All collected teeth had intact roots without caries or periodontitis. The pulp was removed from the crown and the roots under sterile conditions and then cut into 1 mm³ pieces. Tissue fragments were digested in a solution of 3 mg/mL collagenase type I (Solarbio, Beijing, China) and 4 mg/mL dispase (Roche, Basel, Switzerland) for about 60 min and mixed well every 10 min. Single‐cell suspensions were obtained by passing the cells through a 70‐mm strainer (BD Labware, Franklin Lakes, NJ, USA), seeded into 25 cm² culture flask, and maintained in the α‐modification of Eagle's medium (α‐MEM; HyClone, Logan, Utah, USA) containing 20% foetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 0.1 mg/mL penicillin–streptomycin (Biosharp, Hefei, China), which was termed as basal medium. Cells were cultured in an incubator at 37°C in 5% CO2. The culture medium was refreshed every 2–3 days. Passages 3–5 of DPSCs were used in the following studies.