Sensory Neurons Derived From Neural Tissue-committed Stem Cells Of Bone Marrow In Repairing Sensory Defects Of Peripheral Nerve

The compression of the spinal nerve root caused by the reasons of prolapse of lumbarintervertebral disc herniation, lumbar spinal stenosis, lumbar sacral instability and someother reasons can lead to the pain of waist and lower extremities. The clinical patients maybe likely to loss the movement function of their legs in some certain severe cases. Thesensory dysfunction of the affected lower limbs will be never recovered to the normal levelalthough by the applied operations of fenestration decompression, fanestration discectomyand nerve root decompression.The obstacle of pain, temperature sensation and the later ataxia is the mainly clinicalfeatures of the impaired sensory function. In addition, the loss of sensory nerve may beincurred to the vanished of the relevant physiological reflexes. For example, the functiondefect of the pudendal nerve and pelvic splanchnic nerve can lead to the gatism caused bythe disappearing of the nervous reflex. So, we should pay more attention to the patient’ssensory functional recovery with the purpose to improve the life quality of the patients.The transplantation of neural stem cells to repair the nerve defects is an effective andaccepted method.The most important thing is to find a suitable source of the cells for transplantation.The neural tissue committed stem cells (NTCSCs)drived from bone marrow are a subtypeof bone marrow mesenchymal stem cells which can be differentiated into neuron-like cellsnaturally. The advantages of NTCSCs derived from bone marrow were autologous cells,none ethics controversy, immunological privileged and easy accessibility which turn it tobe an ideal source of seed cells for cell transplantation.The second question of the cell transplantaion for repairing the peripheral nervedefects is to find an appropriate animal models for the regenerative medicinetransplantation. The sensory neurons of dorsal root ganglion are in charge of the functionto deliver the peripheral sensory information to the central nervous system. An overdose of pyridoxine through the intraperitoneal injection can caused degeneration and necrosis ofthe nerve axon and neurons of the dorsal root ganglia in the Helgren’s experiment. Thedorsal root ganglion neurons cannot be regenerated because of their characteristic of theterminally differentiated neural cell. This feature results in the abnormalities of sensoryfunctions of the experimental animal model. This experimental method can be duplicatedeasily. So, the sensory nerve defects of animal model made by the method of injecting anoverdose of pyridoxine was decided to carry out in our experimental research.Therefore, we intend to repair the peripheral sensory nerve defects by the means ofthe transplantation of neural tissue-committed stem cells from bone marrow. This can helpus to provide theoretical and experimental foundation for regenerative medicine research inthe treatment of peripheral nerve defects.Part one: The isolation, culture, differentiation and identification ofthe sensory neurons derived from the NTCSCs of bone marrowObjective: To obtain the sensory neurons derived from the NTCSCs of bone marrowwhich can be applied to cell transplantation for the regenerative medicine research.Materials and Methods: Obtained single nuclear cells from bone marrow by thetechnique of cell separation. The screening cell was picked up according to theircharacteristic capacity of the adherent to the bottom of the culture bottle. The screeningcells were amplified in the neurabasal medium by the method of suspension culturewithout serum. We identified the cells harvested from bone marrow, and then induced thecells to differentiate into sensory neurons by the combined application of SHH and RA.We had identified the characteristic of the induced cell in the end.Results: We obtained the neural balls from the bone marrow by the means of singlenuclear cell clone and suspension culture with no serum.The neural balls stem cell expressNestin and CXCR4proteins, and have the similar type features with the MSCs (CD29(97.81%), CD44(91.31%), CD90(93.85%), CD105(95.08%), CD34(3.78%) and CD45(1.59%))which were identified by flow cytometry. Ultrastructure of the cells displaysmultiple nucleoli, abundant mitochondria, euchromatin just like the ordinary stem cellcharacteristics. The induced cells were identified by morphological feature andimmunochemistry specific markers of tissue-committed stem cells and neural stem cell. The experimental results show that the NTCSCs and the bone marrow-derivedmesenchymal stem cells were homologous cells. The induced NTCSCs showed the samecharacteristic with the sensory neurons by the combined induction of SHH and RA. Theinduced cells were proved to express the proteins of NeuN, MAP-2, β-tublinⅢ and GluR-4with the increasing Gria4, Gria3, Calb2, Pou4f1, Tau, NeuN, MAP-2, Tubulin Ⅲ, NF,GFAP, Neurogenin2and descending Nestin, Ret, GATA3genes. The proteins of Tau,MAP-2, NF, NSE, NeuN and Gria4were increased and the Nestin protein was depressed.Conclusion: NTCSCs exists in the bone marrow. They can be obtained by theseparation method and be amplified by suspension culture. NTCSCs can be induced toglutamatergic sensory neurons by the combined induction of SHH and RA.Part two: The establishment of animal model for the sensory defectsof peripheral nerveObjective: To establish a reliable animal model for the sensory defects of peripheralnerve.Materials and methods: The animal model of the sensory defects of peripheral nervewas obtained by the way of intraperitoneal injection pyridoxine hydrochloride（800mg/kg/d）twice a day for8days. To evaluate the result of the model animal, featuresof animal behavior and neural electrophysiology were detected in vivo. The morphologicalfeatures of the injured dorsal root ganglion and sciatic nerve tissue were observed by aseries of experimental methods.Results: The ethology of model animal displayed that the pain and temperaturesensation were defects. The neural electrophysiology displayed that the extended time ofincubation period and the decelerated speed of the sensory nerve conduction velocity. Theneurons of dorsal root ganglion were atrophic, degenerative and necrotic. The nervefibers of sciatic nerve were degeneratived with axonal degeneration, disorders anddistorting, etc.Conclusion: We obtained the reliable animal model for sensory defects of peripheralnerve by the means of injecting an overdose pyridoxine. Part three: The mechanism of sensory neurons derived from neuraltissue-committed stem cells of bone marrow on repairing sensorydefects of peripheral nerveObjective: The research the function and mechanism of the sensory neurons derivedfrom NTCSCs of bone marrow in repairing the sensory defects of peripheral nerve.Materials and Methods: We transplanted the sensory neurons marked with CM-Dilto the L4and L5dorsal root ganglions by the method of micro-injection to repair thesensory defects of peripheral nerve. The result of the behavioral observation, sensoryevaluation and sciatic nerve electrophysiology detection were performed in vivo at the endof2weeks,4weeks,8weeks and12weeks, respectively. The morphological character ofthe dorsal root ganglion and sciatic nerve were observed by microscope and calculated bythe statistical treatment. The repaired sensory nerve pathway was observed by theretrograde tracing reagents-FG.Results: The general posture and the gait of the movement were improved obviously.The temperature and pain sensibility of the animal’s foot were recovered gradually. Themyelinated nerve fiber and sensory nerve conduction velocity of sciatic nerve wereincreased, the incubation period was reduced. These results were linear related with thechange of time. The migration, settled down of the sensory neurons with the CM-Dil tagwere observed by the means of immunofluorescence staining. The result revealed that theneurons expressed the proteins of Neun and GluR-4. The positive cells with FG markedwere detected in the DRG and the cell count increased significantly.Conclusion: The transplantation of sensory neurons derived from NTCSCs of bonemarrow had recovered the sensory defects of the model animals in regenerative medicinesuccessfully.