Isolation of mesenchymal stem cells from dental pulp of exfoliated human deciduous teeth

The exfoliated human deciduous tooth [SHED] contain multipotent stem cells that identified to be a population of highly proliferative and clonogenic .These cells are capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts. Normal exfoliated human deciduous incisors collected from six- to nine-years-old children. The pulp was separated from the crown and digested with collagenase .Single cell solutions were cultivated in alpha-MEM supplemented with ES-FCS. After two to three days, the cells reached confluency and were trypsinized and cultured for further passages. The passage-4 cells were analyzed with CD34, CD45, CD105, CD166, CD31, CD90 and CD146 markers that indicated these cells had a mesenchymal stem cell [MSC] identity. We examined the cells for Alkaline Phosphatase activity to investigate the mesenchymal [stromal] nature.Finally, the cells were differentiated into the osteoblastic and adipocytic lineages in different subcultures and analysed by RT-PCR and different staining protocols. Viable cells growing out of the explants showed elongated shapes in clusters. These cells showed alkaline phosphatase activity. Flow cytometry results revealed high expression of pluripotent stem cell markers .In some area of the osteoinductive cultures nodule-like structures were observed that showed red mineralizing area upon staining with Alizarin Red.In adipogenic cultures lipid vesicles appeared after five weeks of induction with Oil Red. This study show that pulp contains cells with high plasticity and proliferation capacity and can be easily isolated without any serious intervention