Gene Lee

Background: Deciduous tooth pulp derived epithelial stem–like cells (DPESCs) have been reported to be an alternative type of dental epithelial cell that can be used for tooth regeneration. The epithelial cell characteristics and differentiation capacity of rodent odontoblastic cells have been reported previously; however, the ability of these cells to differentiate from human dental stem cells has not been reported. Method: DPESCs were cocultured with dental pulp stem cells from deciduous teeth (dDPSCs) to induce differentiation through epithelial‒mesenchymal interactions. Differentiation patterns were compared between dDPSCs cocultured with DPESCs and dDPSCs alone. To confirm the ability of DPESCs to induce differentiation in dental epithelial cells, induced pluripotent stem cells (iPSCs) were differentiated into dental epithelial cells on a DPESC feeder layer treated with mitomycin C. Established epithelial-like cells (DDes) were characterized and differentiated with dDPSCs. Results: DPESCs cocultured with dDPSCs showed a differentiation pattern with significantly high expression of amelo-/odontogenic differentiation markers such as KLK4 , ENAM , Amelogenin , OCN , DMP1 , and DSPP 8 days after differentiation induction. DSPP protein was also highly expressed in the cocultured DPESCs with dDPSCs. Calcium deposits were observed later than in cocultured DPESCs with dDPSCs than in HERS/ERM cells but were observed in all three DPESC lines during the long differentiation period. DPESC could form a hard tissue structure 12 weeks after in vivo transplantation. iPSCs on the DPESC feeder layer successfully differentiated into epithelial-like cells, and DDes expressed epithelial stem cell markers such as E-cadherin , ABCG2 , EpCAM , Bmi1 , and p75 , as well as stemness-related markers such as Oct-4 , Nanog , and Sox-2 , similar to HERS/ERM cells. In DDes cocultured with dDPSCs, the expression of the KLK4 , ENAM , OCN , DMP1 , and DSPP genes and DSPP protein increased 8 days after differentiation induction, and mineralization was detected 20 days after differentiation induction. Conclusions: The results confirmed the differentiation capacity of DPESCs through epithelial‒mesenchymal interactions and the epithelial differentiation ability of DPESCs. These results suggest that both the epithelial cells and mesenchymal stem cells necessary for tooth regeneration can be secured through the preservation of deciduous teeth.

Research on tooth regeneration using human-induced pluripotent stem cells (hiPSCs) is valuable for autologous dental regeneration. Acquiring mesenchymal and epithelial cells as a resource for dental regeneration is necessary because mesenchymal–epithelial interactions play an essential role in dental development. We reported the establishment of hiPSCs-derived dental epithelial-like cell (EPI-iPSCs), but hiPSCs-derived dental mesenchymal stem cells (MSCs) have not yet been reported. This study was conducted to establish hiPSCs-derived MSCs and to differentiate them into dental cells with EPI-iPSCs. Considering that dental MSCs are derived from the neural crest, hiPSCs were induced to differentiate into MSCs through neural crest formation to acquire the properties of dental MSCs. To differentiate hiPSCs into MSCs through neural crest formation, established hiPSCs were cultured and differentiated with PA6 stromal cells and differentiated hiPSCs formed neurospheres on ultralow-attachment plates. Neurospheres were differentiated into MSCs in serum-supplemented medium. Neural crest-mediated MSCs (NC-MSCs) continuously showed typical MSC morphology and expressed MSC markers. After 8 days of odontogenic induction, the expression levels of odontogenic/mineralization-related genes and dentin sialophosphoprotein (DSPP) proteins were increased in the NC-MSCs alone group in the absence of coculturing with dental epithelial cells. The NC-MSCs and EPI-iPSCs coculture groups showed high expression levels of amelogenesis/odontogenic/mineralization-related genes and DSPP proteins. Furthermore, the NC-MSCs and EPI-iPSCs coculture group yielded calcium deposits earlier than the NC-MSCs alone group. These results indicated that established NC-MSCs from hiPSCs have dental differentiation capacity with dental epithelial cells. In addition, it was confirmed that hiPSCs-derived dental stem cells could be a novel cell source for autologous dental regeneration.

Once tooth development is complete, odontoblasts and their progenitor cells in the dental pulp play a major role in protecting tooth vitality from external stresses. Hence, understanding the homeostasis of the mature pulp populations is just as crucial as understanding that of the young, developing ones for managing age‐related dentinal damage. Here, it is shown that loss of Cpne7 accelerates cellular senescence in odontoblasts due to oxidative stress and DNA damage accumulation. Thus, in Cpne7‐null dental pulp, odontoblast survival is impaired, and aberrant dentin is extensively formed. Intraperitoneal or topical application of CPNE7‐derived functional peptide, however, alleviates the DNA damage accumulation and rescues the pathologic dentin phenotype. Notably, a healthy dentin‐pulp complex lined with metabolically active odontoblasts is observed in 23‐month‐old Cpne7‐overexpressing transgenic mice. Furthermore, physiologic dentin was regenerated in artificial dentinal defects of Cpne7‐overexpressing transgenic mice. Taken together, Cpne7 is indispensable for the maintenance and homeostasis of odontoblasts, while promoting odontoblastic differentiation of the progenitor cells. This research thereby introduces its potential in oral disease‐targeted applications, especially age‐related dental diseases involving dentinal loss.

Backgroud: Research on tooth regeneration using human-induced pluripotent stem cells (hiPSCs) is valuable for autologous dental recovery. Acquiring mesenchymal and epithelial cells as a resource for dental regeneration is necessary because mesenchyme-epithelium interactions play an essential role in dental development. However, the generation of dental mesenchyme from hiPSCs has not yet been reported. Objectives: This study attempts to establish hiPSC-derived mesenchymal stem cells (MSCs) with odontogenic potential. Since most dental MSCs are derived from the neural crest, hiPSCs were induced to differentiate into MSCs through neural crest formation to acquire the properties of dental MSCs. Methods: To differentiate into the neural crest, established human iPSCs were cultured and differentiated on PA6 stromal cells, and differentiated hiPSCs formed neurospheres on ultralow attachment plates. Neurospheres were cultured in serum-supplemented medium and differentiated into MSCs. The properties of neural crest-mediated MSCs (NC-MSCs) were verified by characterization of MSC markers and coculture with Hertwig's epithelial root sheath/epithelial cell rests of Malassez (HERS/ERM). Results: The results showed that NC-MSCs continuously expressed MSC markers for 15 passages. NC-MSCs and HERS/ERM cocultured under odontogenic induction conditions expressed odontogenic/osteogenic-related genes and DSPP proteins and yielded calcium deposits. Under in vivo conditions, NC-MSCs cocultured with HERS/ERM expressed DSPP proteins and formed dentin-like structures after 12 weeks. Conclusion: These results indicated that NC-MSCs established from hiPSCs are a novel cell source for dental recovery.

Tooth development and regeneration occur through reciprocal interactions between epithelial and ectodermal mesenchymal stem cells. However, the current studies on tooth development are limited, since epithelial stem cells are relatively difficult to obtain and maintain. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be alternative options for epithelial cell sources. To differentiate hESCs/hiPSCs into dental epithelial-like stem cells, this study investigated the hypothesis that direct interactions between pluripotent stem cells, such as hESCs or hiPSCs, and Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cell line may induce epithelial differentiation. Epithelial-like stem cells derived from hES (EPI-ES) and hiPSC (EPI-iPSC) had morphological and immunophenotypic characteristics of HERS/ERM cells, as well as similar gene expression. To overcome a rare population and insufficient expansion of primary cells, EPI-iPSC was immortalized with the SV40 large T antigen. The immortalized EPI-iPSC cell line had a normal karyotype, and a short tandem repeat (STR) analysis verified that it was derived from hiPSCs. The EPI-iPSC cell line co-cultured with dental pulp stem cells displayed increased amelogenic and odontogenic gene expression, exhibited higher dentin sialoprotein (DSPP) protein expression, and promoted mineralized nodule formation. These results indicated that the direct co-culture of hESCs/hiPSCs with HERS/ERM successfully established dental epithelial-like stem cells. Moreover, this differentiation protocol could help with understanding the functional roles of cell-to-cell communication and tissue engineering of teeth.

Hyun Nam, Ji-Hye Kim, Ji-Yoon Hwang, Gee-Hye Kim, Jae-Won Kim, Mi Jang, Jong-Ho Lee, Kyungpyo Park, and Gene Lee. Mol. Cells 2018;41:515-22. https://doi.org/10.14348/molcells.2018.0060

Yoon-Kyung Bae, Gee-Hye Kim, Jae Cheoun Lee, Byoung-Moo Seo, Kyeung-Min Joo, Gene Lee, and Hyun Nam. Mol. Cells 2017;40:386-92. https://doi.org/10.14348/molcells.2017.0004

Han JH, et al. J Periodontal Implant Sci. 2015 Apr;45(2):69-75. https://doi.org/10.5051/jpis.2015.45.2.69