Therapeutic potential of HERS spheroids in tooth regeneration.

Hertwig's epithelial root sheath (HERS) plays indispensable roles in tooth root development, including controlling the shape and number of roots, dentin formation, and helping generate the cementum. Based on these characteristics, HERS cell is a potential seed cell type for tooth-related tissue regeneration. However, the application is severely limited by a lack of appropriate culture methods and small cell numbers. Methods: Here, we constructed a 3D culture method to expand functional HERS cells into spheroids, and investigated characteristics and application of dental tissue regeneration of these spheroids. HERS spheroids and HERS cells (2D monolayer culture) were compared in terms of biological characteristics (such as proliferation, self-renewal capacity, and stemness) in vitro and functions (including differentiation potential and inductive ability of dentin formation) both in vitro and in vivo. Further, transcriptome analysis was utilized to reveal the molecular mechanisms of their obvious differences. Results: HERS spheroids showed obvious superiority in biological characteristics and functions compared to 2D monolayers of HERS cells in vitro. In vivo, HERS spheroids generated more mineralized tissue; when combined with dental papilla cells (DPCs), HERS spheroids contributed to dentin-like tissue formation. Moreover, the generation and expansion of HERS spheroids rely to some degree on the HIF-1 pathway. Conclusion: HERS spheroid generation is beneficial for functional HERS cell expansion and can provide a useful cell source for further tooth regeneration and mechanistic research. Notably, HIF-1 pathway plays a critical role in HERS spheroid formation and function.