Culture, Identification And Transplantation In Vivo Of Neural Stem Cells From The Spinal Cord Of Adult Rats

Repair of injured central nervous system (CNS) is always one ofthe highlights of neuroscience. During the latest two decades, studies on neural stem cells (NSCs) come to become the hot-spot of this field. Studies on NSCs, especially the discovery and application of NSCs from the adult mammalian CNS, greatly enrich the theory research of NSCs. The present thesis lays great emphasis on the culture and identification of NSCs from the spinal cord of adult rats and their characteristics in vivo after transplantation.Culture, identify and differentiate NSCs derived from the spinal cord of adult mammals can date back to 1996 at abroad. However, there are still few similar reports at home so far as yet. In the first part of present thesis, NSCs from the spinal cord of adult rats were cultured. Adult male Sprague-Dawley rats were used with body weight ranging between 200-220 g. After anesthesia, one-centimeter-length spinal cord tissue was taken, rinsed with DF12, cut into pieces, and incubated in D-hanks solution containing 0.1% trypsin (Sigma), 0.67 mg/ml hyaluronidase (Sigma) as well as 0.1 mg/ml of deoxyribonuclease I (DNAase I Roche) and aerated with 95% O₂/5% CO₂, at 37 ℃ for 30 min. Then they were neutralized with inhibitor of trypsin, filtered with 70 μm followed by 40 μm nylonmesh sequentially, centrifuged and plated with culture media for NSCs. Under our condition, few small NSCs clones emerged in the culture bottles after plating for 8-10 days. They were congregated with several cells of similar size and smooth cellular edge. The core of these small cell clones bulged with third dimension, which differed from the simple cellular aggregations. Sometimes they are enveloped with the myeline debris in the culture media. The number and volume of these cell clones increased with repeated passages as well as extension of culture time. Usually they are floated in the culture media with few exception of adhesion to the bottom of culture bottle. The cell clones can be observed clearly when cultured for two weeks, some of which still cling to some debris of dead cells or myeline. Immunohistochemical results demonstrated that they strongly express nestin, one of the characteristic markers of NSCs. The properties of these cell clones as NSCs were identified with experiments of self-renewal and differentiation. Similar cell clones emerged with more numbers following mechanical passage in the culture media, suggesting they are amplified in number. These cell clones were passaged as much as 16 generations for three months, and a dramatical number of cell clones were generated, suggesting that they have potent self-renewal capacity. These cell clones were induced to differentiate with either 1% fetal calf serum or suspention from the primary culture media for neurons and astrocytes. Immunohistochemistry demonstrated that the offspring derived from these cell clones express the characteristic markers for neurons,astrocytes and oligodendrocytes, suggesting they are of multipotency. These results above clearly proved that the cell clones we harvested are NSCs indeed.In the second part, these neurospheres were labeled with Hoschet 33342, and then transplanted into the striatum of adult Sprague-Dawley rats on one side. The rats were fixed with perfusion 2 and 4 weeks later, and the whole brains were cut. The distribution of the labeled cells was observed under fluorescence microscope. The transplanted NSCs were found to survive in the brains of adult rats, and gradually migrate into adjacent brain regions such as corpus callosum, subventricular zone (SVZ) of lateral ventricle and cortex. These NSCs migrating into SVZ could integrate into the rostral migratory stream (RMS) futher, and even into the olfactory bulb (OB) in the forebrain. Immunodouble-labeling study showed that population of the NSCs migrating into OB expressed NeuN (one of the characteristic markers of mature neurons) but not GFAP, suggesting they can differentiate into neurons and inhabit in the OB. No labeled NSCs was found in the contralateral striatum, SVZ and OB.The present results show that the NSCs were isolated from the spinal cord of adult rats successfully, which had potent self-renewal capacity and muptipotency. They can survive for at least one month after transplanted into striatum of adult rats, and migrate into the ipsilateral OB in the forebrain down RMS. Some of them can differentiate into neurons in the OB. Our results provide evidence for the culture, genetic modification and transplantation of NSCs in vivo...