Adult human neural stem cells : Properties in vitro and as xenografts in the spinal cord

Abstract: Though the presence of stem cells in the adult human brain has been presented earlier, much has yet to be discovered about these cells. However, the mere potential of these cells has had a significant impact of how we today evaluate the regenerative capacity of the central nervous system and, importantly, on the possible means for science to provide insights in neural repair. In this thesis a series of in vitro studies, based on the formation of neurospheres, was used to identify the subependymal zone and hippocampus as sources of adult human neural stem cells. Cells of hippocampal origin were acquired from patients treated for epilepsy. To access the subventricular zone cell-population, a minimally invasive method was developed: using an endoscope in conjunction to placement of a ventricular drainage catheter in hydrocephalic patients, small biopsies were successfully acquired. Single, candidate stem cells were under strict clonal conditions induced to give rise to neurospheres in the presence of mitogens-epidermal growth factor (EGF) and basic fibroblast growth factor-2 (bFGF-2). The neurospheres were enzymatically passaged. As long as a mitogen was present in the culture medium, the cells continued to form neurospheres without differentiating. After induced differentiation, all 3 phenotypes represented in the CNS were present. Using patchclamp analysis, passive membrane properties of glia and neurons were identified. The development of active membrane properties in neuronal off-spring was documented, finally resulting in INa+-dependent repetetive firing of mature action potentials of individually investigated cells. In a subpopulaton of glutamaterigic neuronal off-spring, evidence of synaptic communication between cells lying in a network was also recorded. Knowing that the neuronal off-spring of adult human neural stem cells was functional in vitro, noncommitted adult human neural stem cells were transplanted into the spinal cord of adult rats in two separate studies. In a future perspective of auto-tranplantation, it is imperative to analyse the behaviour and integrational capacity of these cells, as well as analysing any possible negative side-effect of transplantation therapy using these cells as grafts. We could establish that even after prolonged frozen storage, stem cells survive, migrate and differentiate at least 8 weeks after a xenografting procedure. Migration was more prominent in the rostro-caudal axis and rnicroenvironmental cues seemed to strongly favour astroglial differentiation, although a few neurons of grafted origin also were encountered. A possible devastating side-effect of the procedure of injecting stem cells is the development of a chronic, many times therapy-refractory pain condition called allodynia. In the last study the induction of allodynia by grafted stem cells was evaluated. Using 4 different groups of animals (stem cell injected, vehicle injected, sham laminectomized and intact control), presence of allodynia was evaluated over an 8 week postoperative period. On the ipsilateral side of injection, there is a recovery of stem cell injected animals present after 5 weeks, while the vehicle treated animals showed a delayed response. On the contralateral side of injection however, there is a distinct separation between the 2 injected and the 2 non-injected groups. Importantly, the data here presented does not imply an aggravation of allodynia by injecting stem cells into the spinal cord. The separation in time of recovery between stem cell injected and vehicle treated animals on the ipsilateral side, might suggest a direct effect of the stem cell on host.