The Biological Effects Of BFGD On Human Stem Cells From The Apical Papilla

IntroductionStem cells from the apical papilla (SCAP) are a population ofmesenchymal stem cells (MSCs) residing in the apical papilla of incompletelydeveloped teeth. SCAP are a distinct source of potent dental stem/progenitorcells and appear to have a greater dentinogenic potential than dental pulp stemcells (DPSCs). The distinction between dental pulp and apical papilla is thatapical papilla is the precursor tissue of the radicular pulp. Recently, SCAP havebeen shown to possess the ability to regenerate vascularized human dentalpulp-like tissues in emptied root canal space and produce new dentin-like tissueon existing dentinal walls. These pieces of evidence support the hypothesis thatSCAP may be the source of primary odontoblasts that are responsible for theformation of root dentin.Tooth root formation is mediated through a series of epithelial-mesenchymal interactions regulated by several signaling pathways. Basic fibroblast growth factor (bFGF) is a member of FGFs, which plays an importantrole in morphogenesis and histogenesis of tooth and they effectively promote thetooth root elongation and periodontal tissue formation. More importantly, bFGFwas observed in differentiating odontoblasts at the apical end of the tooth and inthe furcation zone of the developing root at all the stages suggesting that bFGFmay participate in the signaling network associated with root development.bFGF is known an angiogenic and pleiotropic growth factor involved in theproliferation, differentiation and migration of numerous cell types. It is knownbFGF is a critically important factor to maintain self renewal ability of humanembryonic stem (hES) cells in cultures. For adult stem cells, the effects of bFGFon MSCs of different tissue origin are different. bFGF appears to exertdifferential effects on different cell types and the stage of differentiaton of cellsmay respond differently to bFGF as well. Some studies, however, have reportedthat bFGF can up-regulate or down-regulate the ALP activity, promote orsupress the differentiation potential of DPSCs. However, the effects of bFGF onSCAP have not yet been reported. Thus, it is important to investigate the effectsof bFGF on the biologic functions of SCAP in order to understand its regulatoryrole in this context.The purpose of this study was to examine the effects of bFGF on thebehavior of SCAP in terms of cell proliferation, differentiation potential,stemness, and whether MAPK was involved in the differentiation of SCAP bybFGF.Main results1. Isolation of SCAP and characterization of multipotent differentiationability. Human third molars with developing roots were collected due to orthodonticreasons. Procedures were performed according to the approval of theinstitutional review board and the informed consent of the patients. SCAP wereisolated, cultured and expanded as previously described. Cells were collectedand prepared for limiting dilution procedures to obtain single-colony-derivedstrains. Growth characteristics and multipotent differentiation of the cell wereassessed.2. Biological effects of bFGF on SCAP.Different concentration of bFGF all increased the proliferation of SCAP. Theconcentration of5,1020ng/ml were significantly increased SCAP proliferation..bFGF proceeded into the G2/S phase of SCAP. The numbers of CFU-Fformation of SCAP at passages3,6and12were increased with bFGF. Incontrast, bFGF reduced the ALP activity and mineral nodule formation. bFGFdown-regulated the expression of ALP, BSP, DSPP and OCN especially afterSCAP cultured in osteo/dentinogenic medium for2week. SCAP pretreated withbFGF for1week can increase the ability of calcium deposition. bFGFup-regulated the expression of PPAR-γ2after3weeks of adipogenic induced.Regardless of the passage numbers, bFGF increased the percentage of STRO-1+cells significantly and increased the expression levels of pluripotent gene (Oct4,Nanog, Sox2and Rex1) expression levels. These results indicated bFGF canincrease the stemness of SCAP.3. The mechanism of biological effects of bFGF on SCAP differentiationand remain stemness.After SCAP treated with bFGF for different time, the phosphorylation ofJNK, P38and ERK were determined by Western blot assay. bFGF can inducedphos-JNK, phos-ERK and phos-p38expression. The inhibitors U0126, SP600125or SB203580can partly withdraw thesuppression of bFGF on the expression of DSPP, BSP and OCN in SCAP, whichwere cultured in mineralization differentiation media for1week. After treatedwith SP600125，U0126and SB203580respectively, SCAP were cultured instandard medium with or without the presence of bFGF for6days. Theinhibitors only could be partially withdraw the effect of bFGF, Nanog, Oct4andSox2decreased but Rex1not. ERK and JNKpathway play an important role inthe effect of bFGF to SCAP.ConclussionsIn summary, our results suggest that bFGF enhanced the stemness of SCAPevidenced by the increased proliferation, CFU-F forming capacity, andexpression of STRO-1, Nanog, Sox2, Oct4and Rex1, while bFGF suppressedALPase activity, calcified nodule formation and osteo/dentinogenicdifferentiation. MAPKs pathway take part in the effects of bFGF on the biologicfunctions of SCAP. Therefore, in this system, bFGF appears to function as afactor to maintain SCAP stemness, by enhancing self-renewal ability andpreventing cell differentiation. This will facilitate the long-term usage of thesecells for various tissue regeneration applications including pulp/dentinregeneration, bioroot formation and even for neural tissue regeneration.