Dental pulp cell research might open a promising application in tooth tissue regeneration. The aim of this study is to establish a protocol for in vitro culture the human dental pulp stem cells to apply in tissue engineering. Human premolar and impacted third molars were collected and disinfected. Dental pulp fragments were cultured with Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) medium supplemented with 10% Fetal Bovine Serum (FBS). Dental pulp stem cells (DPSCs) were identified using proliferation assay, RT-PCR and flow cytometry. Growth of DPSCs on dentin surface was assessed by MTT assay. The study showed that we successfully isolated, cultured and characterized dental pulp cells by outgrowth method. Cultured population of cells expressed in high level of Oct4, CD146, CD90, CD44. DPSCs proliferated on chemically and mechanically treated dentin surface. This research provides important information and a basis for further investigations to establish dental tissue engineering protocols.