Role Of NFIC In The Differentiation Of Apical Papilla-derived Human Stem Cells

The use of stem cells in tissue engineering strongly extends the range of itsapplications in dental pulp/dentin regeneration or the bioroot for the future. Stem cellsderived from the apical papilla (SCAPs) are a population of mesenchymal stem cells(MSCs) residing in root apex of incompletely developed teeth. SCAPs appear to have acapacity of multi-lineage dentinogenic, especially dentinogenic differentiation when underdefined culture conditions. SCAPs are thought to be the major souce for primaryodontoblasts which are responsible for the formation of root dentin. As a distinct source ofpotent dental stem/progenitor cells, SCAPs show a greater dentinogenic potential andweakly immunogenic compared with dental pulp stem cells (DPSCs), which might be of significance for their application in bone/dental tissue engineering.Nuclear factor I-C (NFIC) was reported as a critical regulator in root formation.Disruption of NFIC caused developed short molar roots contained aberrant odontoblastsand abnormal dentin formation, but normal crowns in mice. Studies have reported thatNFIC played a critical role in root development and SCAPs were the source ofodontoblasts of root. However, the effects and mechanisms of NFIC on the differentiationof SCAPs have not yet been reported.Tooth root formation is mediated through a series of epithelial-mesenchymalinteractions regulated by several factors. The transforming growth factor-β (TGF-β)superfamily, including TGF-βs, bone morphogenetic protein, activins andinhibins,regulates cell proliferation, cell differentiation, the epithelial-to-mesenchymal transitionand embryonic development and so on. Reports have confirmed NFIC affected the normalprogression of the skin wound healing process by inhibiting the level of TGF-β.Disruption of NFIC, both the expression of TGF-β-RI and the phosphorylation of Smad2/3increased during odontoblast, which revealed the interaction between NFIC and TGF-βsignaling pathway. However, the exact mechanism for the regulation and interaction ofNFIC and TGF-β on the differentiation of SCAPs are still unknown.In the present study, we isolated, expanded and identificated SCAPs from the apicalpapilla. The pLenti6.3/v5-NFIC plasmid encoding full-length NFIC or NFIC silence bysi-RNA were constructed and transfected into SCAPs respectively. Then, the effects ofNFIC on the proliferation and differentiation of SCAPs were subsequently determined invitro and in vivo. In addition, we also evaluated the effects of TGF-β1on the proliferativecapacity and differentiation potential of SCAPs. The crosstalk between NFIC and TGF-β1were further determined. These will facilitate the application of SCAPs in pulp/dentinregeneration and bioroot formation.Main results1. Isolation, expandation and identification of SCAPs.Human third molars with developing roots were collected due to orthodontic reasons.The extraction procedure was approved by Institutional Review Board of the Fourth Military Medical University and performed with the informed consent of the patients.Single cell suspensions of SCAPs were obtained by limiting dilution procedures.Immunocytochemical staining and flow cytometry-based cell sorting showed that the cellsderived from apical papilla contained mesenchymal stem cell populations. Besides, theisolated cells had the ability of osteo/odontogenic and adipogenic differentiation, whengrew under defined culture conditions.2. The effects of NFIC on proliferation and differentiation of SCAPs and in vitro.The pLenti6.3/v5-NFIC plasmid encoding full-length NFIC or NFIC silence bysi-RNA were constructed and transfected into SCAPs respectively. The proliferation, ALPactivity and osteo/odontogenic differentiation capacity of SCAPs were increased byoverexpression of NFIC. Besides, NFIC upregulated the mRNA levels of alkalinephosphatase (ALP), osteocalcin (OCN) and collagen typeⅠ(CoL.I), as well as dentinsialoprotein (DSP) protein which were analyzed by western blot. In contrast, knockdownof NFIC blocked the mineralization of SCAPs and downregulated the expression ofodontogenic-related markers ALP, OCN and CoL.I. Additionally, the lipid dropletsformation and the adipogenic differentiation markers (CCAAT/enhancer binding proteinbeta C/EBP-β, CCAAT/enhancer binding protein delta C/EBP-δ and peroxisomeproliferator-activated receptor gamma PPARγ) were further induced by NFIC.Consistently, in vivo results further demonstrated that NFIC promoted the odontoblastor dentin like tissues regeneration integrating with the collagen and (calcined bovinebone，CBB) biomaterials in immunocompromised mice after12weeks.3. Involvement of NFIC in the biological effects of TGF-β1on SCAPsTGF-β1inhibited the prolifiration of SCAPs and mineral nodule formation indose-dependant manner. The mRNA levels of ALP, OCN and CoL.I were alldownregulated by TGF-β1. Results indicated that preincubated with the inhibitors ofTGF-β/Smad signaling (SB431542or SIS3) attenuated the suppressive effect of TGF-β1on the differention of SCAPs. TGF-β1inhibited NFIC protein level. In addition,overexpression of NFIC inhibited the effects of TGF-β1on SCAPs while knockdown of TGF-β1on SCAPs.In summary, results suggested that NFIC enhanced the proliferation and mineralizeddifferentiation of SCAPs both in vitro and in vivo, mechanically, we further found thatNFIC may be a key regulator for the inhibitory effect of TGF-β1in calcified noduleformation and osteo/dentinogenic differentiation. Taken together, current study providedvaluable evidence for the application of NFIC in the pulp/dentin regeneration and biorootformation in the future.