Differentiation Of NSCs Into Schwann-like Cells And Related Signaling Pathways

PrefaceSchwann cells (SCs) are the main neuroglial cells of the PNS. They play essential roles in the regeneration of peripheral nerves after injury. Regeneration mechanism of peripheral nerve injury is very complicated and affected by many factors. Treatment by direct end-end surgical repair is used to minor defects and longer gaps depend on autologous nerve grafts. The therapy of long gap peripheral nerve injuries is clinically limited because of grafts availability and donor-site morbility. Tissue engineering techniques which promote the beneficial endogenous responses to nerve injury could provide an alternative repair strategy. Schwann cells can release neurotrophic factors and play a critical role in peripheral nerve regeneration. Moreover, it has been known for many years that they are capable of sustaining remyelination of CNS axons, and unlike oligodendrocytes, their myelin does not present a target for autoimmune demyelinating diseases. However, the requirement for nerve donor material evokes additional morbility and the production of sufficient numbers of SCs is a prerequisite for applying these cells for grafting.Neural stem cells are pluripotent cells capable of self-renewal and generation of multiple, distinct cell types, including neurons, astrocytes, oligodendrocytes of central nervous system and hemocytes, myocytes of other systems. Neural stem cells are experimentally used for the source of transplantation to the disease models and proper donor-cells for regeneration of central nervous system because these cells have high proliferative activation, long-term cultivation potential, the potential in an undifferentiated state before differentiation into other cells and low immunogenicity. A number of studies have now demonstrated that neurospheres from rat adipose-derived stem cells could differentiate into Schwann cells. However, it is not known up to now whether neural stem cells from central nervous system could be induced into Schwann cells.Mitogenic activated protein kinase pathway (MAPK) plays a crucial role in growth and differentiation. There are three major forms of MAPK pathway in mammals, including extracellular signal regulated kinase (ERKs), c-jun N-terminal kinase (JNKs) and P38 MAPK. ERKs are basic signal pathway in mediation of cell proliferation, differentiation and apoptosis. JNKs and P38 pathway are activated by several conditions, such as environmental stress, cytokine, growth factors. The effects of MAPK in different cell lineages are controversial and it is not known up to now whether MAPK had any effects in the process that neural stem cells differentiated into Schwann cells.In this study, we investigated whether neural stem cells from hippocampus of neonatal rats could differentiate into Schwann cells in the media containing HRG, bFGF, PDGF-AA. We cultured neural stem cells in the media containing inhibitors of ERKs, JNKs and P38 MAPK in order to know the effects of the three MAPK pathways in the differentiated process. It would supply theory and experimental foundation of harvesting large number of Schwann cells and clinical therapy for nerve injury by cell transplantation.MethodsThis study cultured neural stem cells of hippocampus from neonatal rats in the serum-free media. Monoclone was used to purify neural stem cells identified by immunofluorescence staining. The level of S-100 and P75 in the differentiated cells were detected by immunofluorescence staining and western blot. The level of P0, Krox-20 and Oct-6 mRNA in the differentiated cells were evaluated by RT-PCR. The function of differentiated cells was evaluated by lengths of neurites co-cultured with Schwann-like cells. The effects of inhibitors of ERK,JNK,P38 pathways in the differentiation of neural stem cells were detected by western blot.Results1. Culture and identification of neural stem cells of hippocampus of neonatal ratsNeural stem cells dissociated from hippocampus of neonatal rats were suspended growing, neural spheres-like and have intensive light refraction. Passaged cells grew faster than primary culture. The cultured monoclonal spheres expressed Nestin, and expressed NSE, GFAP and Galc one week after induced differentiation.2. Differentiation of neural stem cellsThe shapes of neural stem cells changed in the media containing HRG, RA, FSK, and PDGF-AA. Differentiated cells expressed S-100 and P75 detected by immunofluorescence staining and western blot. The expressions of P0,Krox-20 and Oct-6 mRNA in the Schwann cells and differentiated cells were consistent with the results reported by Marie.3. The effects of proliferation and apoptosis of neural stem cells induced by inhibitors of ERK1/2,P38,JNK pathways in different concentrationThere were no significant differences among the groups that the concentration of inhibitors was 5μM. The cells in P38 group increased gradually and have significant difference with control group. There was necrosis in the cells of central spheres and connected in the flake in P38 group 3 weeks after culture. However, cells in ERK group and JNK group were died 2 weeks after culture. Apoptosis of cells was detected by TUNEL staining. The proportion of apoptosis cells in P38 groups was lower than control group, and that in ERK group and JNK group were higher when the concentration of inhibitors was 10μM. However, there were no significant differences among the four groups that the concentration of inhibitors was 5μM.4. The expression of phosphorylated ERK, phosphorylated P38, phosphorylated JNK and total ERK, total P38, total JNKThe level of phosphorylated ERK of neural stem cells increased gradually and reached the peak 1 hour after adding inducer. Then its level decreased and recovered to normal. The levels of phosphorylated ERK, phosphorylated P38 and phosphorylated JNK were decreased evidently after adding inhibitors and maintained in a normal level for a long time.5. The results of differentiation Schwann-like cells from neural stem cells after adding inhibitorsThe percentage of Schwann-like cells in the ERK group was lower than control group and there was no significant difference between control group and P38 group, JNK group.