Immortalization and characterization of mouse dental papilla cells

Abstract

Mouse dental papilla cells (MDPCs) can be differentiated into component cell types that have the ability to regenerate the pulpo-dentinal complex. At the cap stage of tooth development MDPC component cells are programmed for tooth generation. Therefore, isolated and cultivated component cells can be useful in evaluating cellular, molecular, and environmental scenarios in tooth germ formation and tooth morphogenesis. Since the programmable stage for whole organ regeneration is confined to the brief moment of the cap stage, such cells have a short lifespan in the laboratory. Increase the proliferation capacity of MDPCs and preserve its original phenotypic and genotypic characteristics for tooth organ regeneration. An immortalized mouse dental papilla cell line was created via intracellular insertion of SV40 T antigens into the nucleus by lentivirus particles. The generated clonally-isolated Sv40 T immortalized MDPC line was then characterised and validated for transfection success and efficiency. Detection of SV 40 T cellular expression was carried out using western blotting and RT-PCR protocols. To test for multi potency the cells were forcibly induced into adipogenic, osteogenic and chondrogenic lineages. The immortalized MDPC line showed clear expression of SV40 T antigen both in western blotting and RT-PCR experiments. These cells displayed a higher proliferation rate and both genotype and phenotype characteristics were similar to the original primary cell line. This was highlighted via expression of a broad array of tooth specific markers. Furthermore, the test results to show transformed cells preserved multi potency were also positive. Immortalised cells were inducted into an adipogenic phenotype, as shown by production of lipid globules and an osteogenic phenotype, by forming mineralized nodules. However, the osteogenic marker, ALP was expressed by induced cells from 2 weeks following induction. Established, stable immortalized MDPCs may be used to determine the mechanisms of an array of developmental phenomena such as, early dental cell proliferation, reconstitution of tooth germ, dentine mineralization and other significant growth factor signaling pathways influencing tooth morphogenesis.