Properties of Dental Pulp Stem Cells : Storage Conditions , Longevity , Immortalization , and Senescence

The study of neurogenetic disorders such as Angelman, Rett and Fragile X syndromes as well as autism spectrum disorders has been challenging because of limited testing models. So far, these studies rely on the analysis of gene/protein expression in nonneuronal biospecimens like lymphoblast and fibroblast cell lines. Here we propose to use dental pulps from extracted primary teeth as a source of dental pulp stem cells (DPSCs) for the study of a variety of neurogenetic syndromes. Currently there are no established protocols to successfully obtain DPSC lines from individuals with neurogenetic syndromes. The aim of this study is to examine the effects of storage temperature, time of processing, immortalization, and cellular senescence of DPSCs to determine an optimal protocol for DPSC culture and maintenance to provide a long term and potentially renewable research resource. To accomplish these aims 23 extracted primary teeth were collected from neurotypical subjects from the UTHSC College of Dentistry clinics. The teeth were stored at 4°C and 72°C and processed at 24 and 72 hours and then processed to obtain DPSCs. The DPSCs were cultured and maintained to examine how long they remain viable and how the storage conditions affected culture success. DPSCs that were kept under different storage conditions were immortalized before passaging and after passaging to observe if there is an optimal time for immortalization and whether or not storage conditions affect success. A senescence assay was conducted on 4 samples to determine the longevity of the DPSCs. A culture success rate of 62.5% was obtained with similar failure rates among the different storage temperatures and processing times. All seven attempts at immortalizing samples at passage 2 from groups 1, 2, and 3 were successful. All six attempts at immortalization before passaging were not successful for all groups. Four DPSC lines that underwent senescence testing at each passage all showed similar rates of increased cellular senescence as the number of passages increased. The average senescence rate peaked at 93.6% at passage 13. Two DPSC lines had a significant reduction of their senescence rate after passage 13 possibly indicating neoplastic changes. Our results reveal that storage at 4°C and processing DPSCs within 24 hours may yield a slightly higher rate of growth. DPSCs did not survive the immortalization process before passaging for any group but were successfully immortalized for all groups after going through one passage. Senescence testing revealed that DPSCs approach 100% senescence after about 13 passages but there may be a chance for them to undergo neoplastic transformation, which causes a dramatic lowering in the senescence rate.