Effects Of NF-κb Signaling Pathway On The Proliferation And Committed Differentiation Of Human Stem Cells From Apical Papilla (hSCAPs)

Nuclear factor kappa B is located in the cytoplasm and takes part in many pathological and physiological cellular processes. Diverse studies have demonstrated that NF-κB itself or together with other signaling pathways plays an essential role during the tooth organogenesis and eruption process. The abolition of NF-κB pathway may result in a developmental arrest of teeth. However, little knowledge is available about the effects of NF-κB pathway on the proliferation and differentiation of human stem cells from apical papilla (hSCAPs).Part I. Culture and identification of stem cells from the apical papillaObjective:To obtain the purified stem cells from the apical papilla.Methods:Healthy impacted third molars with immature apex were collected. Root apical papillae were carefully isolated from the immature root apexes. Primary apical papilla cells were enzymatically separated as previously described. Then, the primary cells were purified by the method of magnetic activated cell sorting. To determine the nature of cultured cells, immunofluorescence cytochemistry and flow cytometric analysis of specific surface antigens were conducted.Results:The hSCAPs appeared as elongated or fibroblast-like cells. These hSCAPs were stained positively for STRO-1, but negatively for cytokeratin. There was a high expression of mesenchymal stem cell (MSC) markers (e.g., CD29, CD73, CD90, CD105and CD146), while the hematopoietic markers (e.g., CD34and CD45) were expressed at a low level in hSCAPs as demonstrated by flow cytometry.Conclusions:The hSCAPs can be purified by enzyme digestion and magnetic activated cell sorting. These isolated cells were stem cells of mesenchymal origin with high proliferation activity. Part II. Activation and inhibition of canonical NF-κB signaling pathway in hSCAPsObjective:To explore whether hSCAPs treated with TNF-a or BMS-345541can result in the NF-κB activation or inhibition respectively.Methods:hSCAPs from the third-fifth passages were cultured in serum-free media for24hours, followed by the treatment of the putative NF-κB pathway activator TNF-a (10ng/mL) or1μmol/L BMS-345541, the selective inhibitor of IKK. Then the cytoplasmic proteins were extracted and subjected to western blot assay in order to measure the expression of NF-κB pathway-related proteins (IκBα, P-IκBα, P65, P-P65).Results:Phospho-IκBa and phospho-P65were obviously elevated in a time-dependent manner after TNF-a treatment. Suppressed NF-κB activity was detected in hSCAPs after incubation with BMS-345541, as indicated by the decreased phosphorylated IκBα and P65. Quantitatively, ratios of phosphorylated to unphosphorylated forms of proteins at15min-.30min and60min were higher than that at0min in TNF-a-treated hSCAPs, while ratios of phosphor-IκBα to IκBα and phosphor-P65to P65at15min,30min and60min were significantly lower than that at0min in hSCAPs after incubation with BMS-345541.Conclusions:hSCAPs treated with10ng/mL TNF-a or1umol/L BMS-345541can activate or suppress the NF-κB pathway successfully.Part III. Effects of NF-κB pathway on the proliferation and odonto/osteogenicdifferentiaton of hSCAPsObjective:To investigate the effects of NF-κB pathway on the proliferation and odonto/osteogenic potential of hSCAPs.Methods:hSCAPs were cultured in the complete media or mineralization media (MM), with or without the activator or inhibitor. The proliferation of hSCAPs was examined by MTT assay and flow cytometry (FCM). The wound healing assay was conducted to explore the migration ability of hSCAPs. ALP activity, alizarin stain and quantitative calcium measurement were used for the evaluation of osteogenic differentiation. The expression of odonto/osteoblastic markers (ALP、OCN/OCN、 BSP/BSP、OSX/OSX、RUNX2/RUNX2、DSPP/DSP、OPMOPN, and DMP-1/DMP-1) after the activation and inhibition of NF-κB signaling pathway in hSCAPs was measured by real-time RT-PCR and western blot.Results:NF-κB pathway-activated hSCAPs presented a higher proliferation activity as compared with control groups, and flow cytometry assay (FCM) demonstrated that the proliferation index was significantly higher than that in control group. Mineralization experiments in vitro revealed that NF-κB pathway-activated hSCAPs presented the enhanced alkaline phosphatase (ALP) activity, the higher density of calcification nodules and the up-regulated expression of odonto/osteogenic markers (ALP, OCN/OCN, OSX/OSX, BSP/BSP, RUNX2/RUNX2, OPN/OPN, DSPP/DSP, DMP-1/DMP-1). However, NF-κB pathway-inhibited hSCAPs exhibited a lower proliferation, a delayed migration pace, the shorter migration distance and decreased odonto/osteogenic ability in comparison with control groups.Conclusions:NF-κB pathway plays a paramount role in the proliferation and committed differentiation of hSCAPs.