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Title: Neural stem (NS) cells, from mouse, rat, and human
Author: Sun, Yirui
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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Neural stem cells are the self-renewing and oligopotent cell population that generate constituent cell types of the nervous system. Cultured neural stem cells would offer researchers accessible opportunities to answer fundamental questions in both neurodevelopment and cell biology. Current strategies of maintaining neural stem/progenitor cells in vitro largely rely on neurosphere cultures (Reynolds and Weiss, 1992) and/or genetic immortalization (Frederiksen et al., 1988; Sah et al., 1997), These approaches raise concerns about cellular heterogeneity and potential cell transformation. Our lab has recently reported the establishment of adherent mouse Neural Stem (NS) cell lines that undergo symmetrical self-renewal without genetic immortalization (Conti et at., 2005; Pollard et al., 2006). Here, I apply this approach to human and rat foetal tissue and describe the derivation and characterization of human and rat NS cell lines. I established Human foetal NS cell lines from elective termination tissue. Human NS cells are propagated as stable cell lines in the presence of both epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), under which conditions they stably express neural precursor markers and exhibit negligible differentiation into neurons or glia. Human NS cells are induced to produce astrocytes, oligodendrocytes, and mature neurons upon exposure to appropriate differentiating conditions. Human NS cells are clonogenic stem cells. They are capable of generating clonal and tripotent cell lines from single deposited cells, demonstrating they represent self-renewing in vitro human neural stem cell populations. More importantly, human NS cells retain a diploid karyotype and constant neurogenic capacity for more than 100 generations, and their long-term stability does not require leukemia inhibitory factor (LW). Together with the demonstrations that human NS cells can be genetic modified and are accessible to multi-well time-lapse videomicroscopy, these cells creates the potential for high content genetic and chemical screens. In addition to human foetal tissue, adherent NS cells can also be derived from rat foetal brain and spinal cord. However, under standard expansion conditions supplemented with EGF and FGF2 (Conti et al. 2005), rat NS cells spontaneously become dormant after approximately 2 months expansion. Dormant rat cells exhibit stellate morphology and express the astroglial maker GFAP, but they still retain neural precursor makers such as Nestin and Sox2. I found that Bone Morphogenetic Protein (BMP) signals are responsible for generating quiescence of rat NS cells, and that FGF2 signaling inhibits BMP-induced astrocyte differentiation and therefore maintains stem cell potency. Applying NS cell conditioned medium or BMP antagonist Noggin could overcome cell quiescence, and by these means the long-term propagation of rat foetal NS cells can be maintained. In addition to foetal NS cells, Noggin also promotes the proliferation of adult rat subventricular zone (SVZ) neural precursors. These observations implies that the neurogenic but quiescent rat NS cells generated by BMP and FGF2 signals may reflect some characteristics of in vivo adult neural stem cells. Lastly, I undertake preliminary investigation of intracerebral transplantation using established NS cell lines. Mouse NS cells labelled with green fluorescent protein (GFP) were injected into cortex, striatum and hippocampus of both adult and neonatal mouse brain. I find NS cells can survive for at least 6 weeks after transplantation, although their migration appears limited. In adult brain, mouse NS cells differentiate into both astrocytes and morphological neurons expressing interneuron markers including Calretinin and Somatostatin. However, injected cells largely generate astrocyte in neonatal brain. These observations demonstrate that NS cells can be used as donor cells for transplantation studies. Future studies are required to evaluate how human and rat NS cell will behave after transplantation. It would also be informative to investigate whether cultured NS cells may contribute to functional repair in disease models.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available