Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Introduction: Stem cells of the apical papilla (SCAP) have been identified as an important population of mesenchymal stem cells (MSCs) in regenerative endodontics. Preclinical studies that evaluate the various aspects of the regenerative process must use fully characterized MSCs. The phenotype of these cells when maintained in culture is crucial for the translational applicability of these studies. Thus, in this study, we aimed to characterize a SCAP cell line that preferentially expressed and maintained the required MSCs markers in culture, namely the RP-89 cell line. Methods: Apical papillae from extracted mandibular third molars from a single donor were processed for immunohistochemistry, cell culture, and,RT-PCR. SCAP were successfully cultured and maintained in culture for up to 20 passages. The expression of MSC-related molecular markers was analyzed by laser confocal microscopy, flow cytometry, and real-time RT-PCR arrays. Results: Cells within the apical papillae tissue had a widespread expression of CD90, whereas the expression of CD105 and CD73 was compartmentalized to the blood vessels and periphery, respectively. The RP-89 cell line population coexpressed CD73, CD90, and CD105 in all passages evaluated. There was a dramatic change in gene expression when cells were cultured and maintained in culture with the up-regulation of MSCs markers, inhibitors of differentiation, and sternness markers. Conversely, genes involved in the differentiation of MSCs were suppressed. Conclusions: Collectively, these results highlight the need to use fully characterized cell lines in regenerative studies and provide the foundational knowledge of gene expression modulation that occurs in cultured SCAP. (J Endod 2013;39:357-363)393357363University of Texas Health Science Center at San AntonioDepartment of EndodonticsCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPES [BEX3563/103]