Establishment of immortalized dental follicle cells for generating periodontal ligament in vivo

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Abstract
The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, viz., cementum, periodontal ligament (PDL), and alveolar bone, are created by dental follicle progenitors. Here, we report the presence of PDL progenitors in mouse dental follicle (MDF) cells. MDF cells were obtained from mouse incisor tooth germs and immortalized by the expression of a mutant human papilloma virus type 16 E6 gene lacking the PDZ-domain-binding motif. MDF cells expressing the mutant E6 gene (MDF E6-EGFP cells) had an extended life span, beyond 150 population doublings (PD). In contrast, normal MDF cells failed to proliferate beyond 10 PD. MDF E6-EGFP cells expressed tendon/ligament phenotype-related genes such as Scleraxis (Scx), growth and differentiation factor-5, EphA4, Six-1, and type I collagen. In addition, the expression of periostin was observed. To elucidate the differentiation capacity of MDF E6-EGFP cells in vivo, the cells were transplanted into severe combined immunodeficiency mice. At 4 weeks, MDF E6-EGFP cell transplants had the capacity to generate a PDL-like tissue that expressed periostin, Scx, and type XII collagen and the fibrillar assembly of type I collagen. Our findings suggest that MDF E6-EGFP cells can act as PDL progenitors, and that these cells may be a useful research tool for studying PDL formation and for developing regeneration therapies.