Objective To isolate and identify human periodontal ligament stem cells（PDLSC） by improved methods and assess the characteristics of PDLSC ex vivo. Methods The periodontal ligament cells were obtained from the healthy impacted third molars and teeth extracted for orthodontic purposes and used to isolate PDLSC by limiting dilution assay. PDLSC were cultured and expanded in α-MEM supplemented with 10% FBS. Colony-forming assay, immunohistochemistry, flow cytometry, osteogenic and adipogenic induction were used to identify PDLSC. Results The obtained cells had high colony-forming efficiency and were positive staining for vimentin and negative for pancytokeratin. Flow cytometry revealed that the isolated cells were positive for STRO-1 and CD146 antibodies and most were in the G0/G1 phase of cell cycle. Under specific conditions, they could differentiate to the osteoblast and adipocyte lineages in vitro. Conclusion Limiting dilution assay is an effective method to isolate PDLSC and the single- cell-derived colonies demonstrate the properties of stem cells in vitro.
