Neuronal and glial differentiation of expanded neural stem and progenitor cells; in vitro and after transplantation

Abstract: In this thesis we have used cells dissected from the lateral ganglionic eminence (LGE), the medial ganglionic eminence (MGE), and the cortical primordium of the embryonic mouse forebrain. The tissue was dissected from either i) wild-type mice, ii) green fluorescent protein (GFP)-, or iii) Gtv-a-expressing transgenic mice, and subsequently grown and expanded in vitro using two different protocols. Cells were either plated and extensively expanded as attached glial cultures in the presence of epidermal growth factor (EGF) and serum, or expanded in the presence of EGF and basic fibroblast growth factor (bFGF) as free-floating aggregates termed neurospheres. The attached LGE-derived cells were expanded for more that 7 months (25 passages), and the cells expressed neural stem-/progenitor markers, such as glial fibrillary acidic protein (GFAP), nestin, RC2 and M2/M6, both at early and late passages. We demonstrated that the repeatedly passaged attached glial cultures derived from either the LGE or MGE (but not the cortical primordium) were capable of generating significant numbers of neurons and glial cells at differentiating conditions, i.e. after removal of EGF and serum from the expansion medium. By using a transgenic approach, we were able to show that at least a subset of the newly generated neurons and oligodendrocytes were derived from GFAP-expressing cells. Interestingly, the newly generated neurons were found to retain some of their region-specific expression even after extensive in vitro-expansion. After grafting of the expanded attached LGE-derived cells, we found that they were able to integrate into both the adult (intact and lesioned) and neonatal rat striatum, as visualized with the mouse-specific astroglial marker M2. However, even though these cells had the capacity to differentiate into neurons and glial cells in vitro, we were only able to detect few neuron-like cells after transplantation. Instead these cells expressed almost exclusively an astroglial phenotype after implantation. Moreover, we showed that cells from expanded neurosphere cultures, derived from the LGE, MGE and cortical primordium of the embryonic GFP-transgenic mouse, had the capacity to differentiate into morphologically fully mature neurons, as well as astrocytes and oligodendrocytes after transplantation, as visualized with the species-specific marker M2 and the reporter gene GFP. These results demonstrated the ability of mouse derived neural stem-/progenitor cells expanded in vitro as neurospheres to generate both neurons and glia after transplantation into neonatal recipients, and differentiate into mature neurons with morphological features characteristic for each target site. Altogether, the results of the present thesis demonstrate a capacity of cells derived from the mouse embryonic forebrain to be long-term expanded using two different protocols, and that the cells have the potential to differentiate in vitro and give rise to progeny with at least some region-specific characteristics retained. The cells can also survive and integrate into the host tissue after transplantation. However, mainly cells grown as neurospheres displayed the potential of neuronal differentiation after implantation into the neonatal graft host. A lot of experimental work is still needed in order to understand and control the mechanisms for growth and differentiation of neural stem-/progenitor cells before such cells can be applied in other studies, such as in clinical trials towards treatment of for example neurodegenerative disorders.