Study The Cultivation And Differentiation Of DRG-drived Neural Stem Cells

Peripheral nerve injuies are common and results in incomplete or no functional reeovery. The development of tissue engineering has provided a new strategy for the reparation of damaged nerves,the seed cells are critical for the construction of tissue engineering nerve. Schwann cell seems the candidate cell in the celltransplantation while it is difficult to preserve. It is necessity to looking for other cells more fit the transplantation continuously. The trunk neural crest stem cell can differentiate into neuron and glia which contribute to PNS. It is considered as the primordium.The differentiation of stem cell has pleiotropia and indeterminateness because being regulated by lots of factors in vitro or in vivo. It is necessity to induce stem cell to differentiate directly into special cells which were we needed when they are used in treating the diseases of never system. Basic fibroblast growth factor (bFGF) can stimulate stem cell to differentiate into neuron and glia due to it’s multifunctions, and the action is concided with the course of never system development, that is mean that it stimulate stem cell to differentiate into neuron at the early stage of development, while to differentiate into glia at the late stage. In peripheral nervous system, bFGF is the forward directed determinative factor lead the stem cell to differentiated into schwann cell progenitor. So, it is an instructed attempt to drive the trunk neural crest stem cell to differentiate into schwann cell using bFGF. In the paper, using cultured adult rat dorsal ganglia neural crest stem cell (DRG-NCSCs) as a model, we induced DRG-NCSCs differentiate along schwann cells by bFGF, and search a mechanism of the cells differentiation. PartⅠIsolation and character of neural crest stem cell drived from dorsal ganglia and induced directly them to differentiated along schwann cellsObjective:To search the seed cell for peripheral nerve injuies, we approach the neural crest stem cells cultural method in vitro from the rat dorsal ganglia, and to explore the method by which can induce neural stem cells from rat dorsal ganglia to differentiate directly along schwanns cells in vitro.Methods:1. Adopt the SD rat dorsal ganglia,obtained the primary neuropheres, then the singal clony experiments were proceeded by unlimited dilution, and were verificated after passaged three generations by NCSCs specially markers, mitotically active and multi-differentiated potentiality.2. Differentiated the clony cell spheres with NGF, bFGF, NRG, FBS or not. The appearance of differentiated cells and verified the character by immunostaining and western blot with S100 after 7 d.3. bFGF act on the DRG-NCSCs at 24 h or 7d, The appearance of differentiated cells and verified the character by immunostaining and western blot with S100 after 7 d.4. The cell proliferation detected by BrdU labeling in 24h, and meanwhile carried out immunostaining.5. The differentiated cells were characterizated by immunofluorescent cytochemistry, passaged culture, and ensheathment and myelin experiment in vitro. And investigated the protein expression of SCs and differentiated cellsResults:1. The DRG-NSCs which could proliferate stabilized in vitro were obtained from the dorsal ganglia and expressed NCSCs-specific marker; they could produce three kinds of neural cells when being induced to differentiate in vitro.2. Using the cells as a model, we founded that bFGF stimulation caused DRG-NCSCs differentiate along schwann cells, and no effect on cell proliferation within 24 h.3. The obtained cell had the property of schwann cells, such as expressed and secreted neurotrophic factors, taked part in myelin sheath formation in vitro , had homoplastic protein expressed spectra as schwann cell.Conclusions:The experiment results demonstrated that we had obtained the seed cells for for peripheral nerve injuies by finding the adult rat dorsal ganglia neural crest stem cells cultured method and induce them to differentiate directly along schwanns cells in vitro.PartⅡThe mechanisms of the DRG-NCSCs differentiated along schwann cells by bFGFThe functions of bFGF is achieved by binding to the fibroblast growth factor receptors (FGFRs), actived the MAPK,PI3K,PLCγpathways and immediate early genes or transcription. There are at least four structurally related high affinity FGFRs, and the downstream signaling pathways are also varied when bFGF binded with various FGFR.Objective:To investigate the mechanisms of bFGF-stimulation the DRG-NCSCs differentiated along schwann cells by studing the FGFR , the sign pathways and immediate early genes or transcription. Methods:1. RT-PCR and westemblotting we first analyzed the expression of various FGFRs in r DRG-NCSCs, and Cross-linking Experiment and immunoprecipitated detected the receptor which bFGF binded.2. The changes of the activity of MAPK, PI3K, and PLC induced by bFGF or not were accessed by westemblotting.3. The marker expression of the differentiated cells were assessed when added or not the inhibitor which blocked the MAPK, PI3K pathway by westemblotting and irnmunofluorescence technique.4. The activity of immediate early genes were detected by TransAMTM Assay kits. The mRNA expression of transcriptional factor, such as Mash-1 and Sox10 were determined by RT-PCR.Results:1. RT-PCR and Western blot experiments showed that the level of receptor-1was high and levels of receptor-2 and -3 were relatively weak. Results from the chemical cross-linking and immunoprecipitation experiments showed that bFGF mainly bound to the FGFR-1 under our experimental conditions.2. The results of westemblotting showed that the activity of ERK1/2 and PI3K were sustained during the bFGF-stimulated period in contrasted with the control. 3. The results of mass-spectrum, western blot and transcription activation domain assay had proved that bFGF-stimulation caused DRG-NSCs differentiate along schwann cells through an ERK1/2-dependent pathway without influencing the proliferation and survival of differentiated cells within 24 hours. bFGF induced phosphorylation of ERK/2 or AKT, increase the transcription activator of c-Jun and decrease of the expression of Mash-1, a transcriptional factor known to participate never system development. Application of U0126, a specific inhibitor of MEK1/2 which are upstream to ERK1/2, suppressed the the transcription activator of c-Jun and prevented DRG-NSCs differentiation in response to bFGF. However, administration of LY294002, an inhibitor of PI3K, did not affect the effect of bFGF on the the transcription activator of c-Jun and DRG-NSCs differentiation. When bFGF acted on the DRG-NSCs differentiation surpass 24 hours, bFGF influence the proliferation and survival of differentiated cells through an ERK1/2 and PI3K/AKT pathways.Conclusions:bFGF induced DRG-NSCs to differentiate along schwanns cell by binding FGFR1, leading to ERK1/2 phosphorylation, enhancing the transcription activator of c-Jun, and decreasing the expression of Mash-1.