Bone marrow-mesenchymal stem cells as a suitable model for assessment of environmental pollution

Stem cells are considered as ideal model for assessment of environmental toxins on proliferation, multipotency and differentiation. The aim of this study was to investigate the effect of lead as harmaful environmental pollutant on proliferation and neural differentiation of murine bone marrow-MSCs. In this experimental study, MSC cells were exposed to different concentrations of lead [0 to 100 micro M] for 24h, and the level of cell proliferation was determined by 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-dipheny-2H-tetrazolium bromide [MTT] reduction assay. In addition, DNA fragmentation was evaluated with comet assay at a single cell level. To induce the neural phenotype, MSCs were cultured for 2 days in the presence of 50 micro Pb[2+] for 48 h. At the end of this period, the medium was replaced by fresh medium supplemented with 1 mM beta-ME for 24 hr and then fresh medium supplemented with 7 mM beta-ME for 4 hours respectively. The expression of neural marker such as nestin, MAP2, and tau was assessed by immunocytochemistry, while the expression of neuronal specific genes such as Neur-1, Nestin, and beta-tubulin III was determined by RT-PCR analysis. Exposure to lead reduced the level of cell proliferation in a dose-dependent manner. The comet assay of cells exposed to lead showed varying degrees of DNA damaged. Change in cell morphology was observed 1 to 4 hr post neural exposure. The percentage of the MAP2 positive cells was reduced significantly at greater than 40 micro M lead concentration. This observation was further verified by assessment of the expression of neural markers. This study clearly indicated lead is highly cytotoxic to MSCs and these cells appear to be an excellent choice for establishment of guidelines for environmental hazards and drugs on cell proliferation and differentiation

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