Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline‐inducible expression system

Human dental pulp stem cells are a potentially useful resource for cell‐based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user‐friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin‐dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet‐off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC‐K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet‐off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet‐off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet‐off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells. Significance Statement We have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin‐dependent kinase 4 (CDK4R24C) and Cyclin D1 (hereafter referred to as Tet‐off K4DT). In Tet‐off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Tet‐off K4DT cells have demonstrated an extended cellular lifespan, increased proliferative capacity, and had no observable genomic aberrations and displayed a sustained expression of stem cell markers even at relatively advanced passages.