Culture and Molecular Markers Characterization of Stem Cells from Human Deciduous (SHED) and Permanent (DPSC) Teeth Pulp

Human dental pulp stem cells are adult multipotent stem cells isolated from dental pulp tissue. Our objective was to determine in vitro culture technique for stem cells from deciduous tooth (SHED) and permanent tooth (DPSC) through cell passage identification, the effect of trypsin-EDTA and proliferation potential, and to characterize both cells using molecular markers profile. Enzyme digestion method was used on dental pulp tissue from deciduous and permanent tooth for SHED and DPSC isolation, respectively. Both cells were cultured at passage 1 until 5 and trypan blue assay was used to obtain growth curve in determining cell population doubling time (PDT) for each passage. Effect of trypsin-EDTA on both cells were studied using Alamar blue assay to determine optimum incubation time for subculturing process. Cell proliferation potential for both cells within 21 days was determined using 3-(4,5-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (AITT) assay. Cell characterization through molecular biology markers was performed using RT-PCR. Both cells at all passages appeared fibroblast-like. SHED and DPSC at passage 3 exhibited the lowest PDT with 43 +/- 2.3 and 63 +/- 3.1 h, respectively. SHED exposed to nypsin-EDTA showed decrease in cell viability percentage compared to DPSC. Cell growth for SHED was similar to 2.3-fold higher than DPSC. Both cells expressed mesenchymal stem cell markers and not hematopoietic stem cell markers. In conclusion, homogenous morphology and lowest PDT value indicated that cells at passage 3 are the best to determine proliferation potential and molecular markers expression. SHED proliferated better than DPSC. However DPSC was more resistant to trypsin-EDTA than SHED. Based on molecular marker profile, both cells are mesenchymal stem cells.