The long-term goal of this proposal is to identify, isolate, and characterize human adult odontogenetic cell stem lines that are capable of regenerating mature tooth organs. In vitro and in vivo studies have shown that embryonic and adult pulp contain mesenchymal stem cells with the potential to form bone, cartilage and dentin. Recent in vivo studies have suggested that developing human teeth contain cells with the ability to regenerate entire tooth structure. Based on these findings we believe that the human tooth contains stem cell populations that can be induced to reform the entire tooth structure. The purpose of this application is to provide the necessary pilot studies to identify and characterize human tooth stem cells. Our hypothesis is that human tooth organs contain stem cell populations, both mesenchymal and epithelial, that are capable of total tooth tissue regeneration. This application is based on our extensive experience establishing dental pulp mesenchyme and enamel oral epithelium cell lines from primary cell cultures derived from mouse and human tooth tissue explants. These established immortalized dental cell lines are being used to understand the molecular mechanisms controlling tooth-specific gene expression, extracellular matrix formation and biomineralization, cell cytodifferentiation, reparative/tertiary dentin formation, and tooth regeneration. Based on the analysis of these cell lines in relationship to the extensive emerging stem cell literature, we will perform preliminary studies to test our hypothesis that developing teeth contain both mesenchymal and epithelial stem cell populations that when combined are capable of tooth organ regeneration. To test this hypothesis, two specific aims are proposed: 1. To isolate, identify and immortalize human odontogenetic (mesenchymal and epithelial) stem cell populations based on the emerging stem cell literature. 2. To characterize in vitro and in vivo the multipotent potential of these established odontogenetic stem cell lines to differentiate into various dental tissue phenotypes. Currently, no stable human odontogenetic stem cell lines exist and therefore progress in understanding the cell biology, biochemistry, development, and molecular biology of tooth formation and regeneration has been impeded. Cell lines resulting from this work will represent the only available clonal human stem cell lines. Identification, isolation and immortalization of such cell populations would allow detailed biochemical and molecular studies to be conducted on stable, homogeneous populations of cells. This study is innovative and of high risk but with the potential of significant impact on future tissue engineering and biomimetic approaches to clinical dentistry. Our hypothesis requires "proof-of-concept" studies to be performed that will allow future funding through more traditional R01 grant mechanisms.