OBJECTIVETo isolate and identify human dental pulp stem cells from third molars.

METHODSDental pulps were dissected and digested by collagenase type I and dispase. The obtained single cell supernatant were harvested and cultured. Characterization of the phenotype of DPSCs was detected by immunohistochemical method and RT-PCR assay. Cell cycle was analyzed by FCM. Differentiation potential of DPSCs was evaluated.

RESULTSColony-forming efficiency of cells derived from dental pulp tissue was 2 - 15 clones/10(3) cells plated. DPSCs were found to express many different markers, including vimentin, collagen type I, GFAP, nestin and osteocalcin, while they failed to react with MyoD and DSPP. About 64.1% of the cells were in G0/G1 phases, while only 35.8% in proliferation (S + G2 + M). Grown in an adipogenic cocktail medium for three weeks, some DPSCs expressed fat cell markers of PPARgamma and LPL, and formed oil red O-positive lipid clusters in five weeks. After culture with a myogenic-inductive medium, DPSCs were found to express MyoD, desmin and myosin, markers of myocyte. Long-term cultures of DPSCs grown in differentiation inductive medium demonstrated the capacity to form Von Kossa-positive condensed nodules with high levels of calcium.

CONCLUSIONCells isolated from adult human dental pulp are clonogenic, and have multipotent differentiation potential, satisfying the criteria of postnatal somatic stem cell.