Abstract
Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies.