published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

No description available.

Neural stem cell (NSC) transplantation strategy offers great potential to treat spinal cord injury (SCI). NSCs may replace lost neurons or oligodendrocytes, and act as a source of neurotrophic factors to support the survival of remaining cells. Their efficiency was limited by poor survival after transplantation, and they had more tendencies to differentiate into astrocytes, but not neurons and oligodendrocytes. This project investigated whether activated microglia is a factor that contributes to this phenomenon, and studied the potential role of minocycline, a widely used antibiotic drug, to modify the negative effects of microglia on NSCs. In the first part of this study, we used organotypic spinal cord slice (SCS) culture to mimic in vivo local environment after SCI, and NSCs were grafted on their surface or shared culture medium with them. After specific depletion of microglia with clodronate loaded liposome, more grafted NSCs survived, and in the co-culture system, the NSC neuronal differentiation rate increased while glial differentiation rate decreased, the apoptosis rate also decreased. This suggested that activated microglia may impair NSC survival, and neuronal differentiation, but improve glial differentiation. In the second part of this study, we first tested the direct effects of minocycline on NSC apoptosis, proliferation and differentiation in vitro, to test whether minocycline has any side effect on NSCs. The results showed that at the concentration 10μg/ml or lower, minocycline did not affect NSC survival and proliferation, but impaired neuronal differentiation. Then we treated primary microglia culture with LPS or LPS plus minocycline, and collected the conditioned mediums (CM-LPS and CM-LPSMC) to test their effects on NSC　apoptosis and differentiation. The results showed that compared with CM-LPS, CM-LPSMC resulted in a significantly lower apoptotic rate of NSCs, also allowed NSC neuronal differentiation. This suggested that minocycline may impair the pro-apoptotic effect of activated microglia on NSCs. In conclusion, our study showed that activated microglia may impair NSC survival and neuronal differentiation. This indicated that in NSC transplantation strategy for SCI, microglia would be a target to be manipulated to improve graft survival and neuronal differentiation. Although minocycline may suppress NSC differentiation towards neurons, it has the potential to protect NSCs from the toxic effects of activated microglia. This showed the therapeutic potential of minocycline in NSC transplantation strategies for SCI. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 3, 2009) Vita. Includes bibliographical references.

Neural stem cells (NSCs) in the adult forebrain are thought to comprise a subpopulation of cells that express glial fibrillary acidic protein (GFAP), termed B cells. These GFAP+ cells generate proliferating neuroblasts that migrate from the lateral ventricle subependyma along the rostral migratory stream to become olfactory bulb interneurons. Based on this lineage, we set out to create a NSC deficient mouse through targeted ablation of dividing GFAP+ cells in vivo. We successfully depleted the GFAP+ cells as seen using an in vitro colony forming assay in multiple kill paradigms, however we were unable to permanently eliminate the multipotent, self-renewing colony forming cells. Instead, the targeted ablation of GFAP+ cells revealed an upstream, GFAP- cell that was induced to proliferate in the presence of leukemia inhibitory factor (LIF). These findings support the hypothesis that a population of GFAP-, LIF responsive cells are the definitive adult NSC upstream of GFAP+ cells.

Neural stem cells (NSCs) in the adult forebrain are thought to comprise a subpopulation of cells that express glial fibrillary acidic protein (GFAP), termed B cells. These GFAP+ cells generate proliferating neuroblasts that migrate from the lateral ventricle subependyma along the rostral migratory stream to become olfactory bulb interneurons. Based on this lineage, we set out to create a NSC deficient mouse through targeted ablation of dividing GFAP+ cells in vivo. We successfully depleted the GFAP+ cells as seen using an in vitro colony forming assay in multiple kill paradigms, however we were unable to permanently eliminate the multipotent, self-renewing colony forming cells. Instead, the targeted ablation of GFAP+ cells revealed an upstream, GFAP- cell that was induced to proliferate in the presence of leukemia inhibitory factor (LIF). These findings support the hypothesis that a population of GFAP-, LIF responsive cells are the definitive adult NSC upstream of GFAP+ cells.

Neural stem cells (NSCs) are defined as multipotent stem cells. They are able to self-renew and differentiate into mature cells, such as neurons, oligodendrocytes and astrocytes. Neurotoxicity of lead (Pb2+) has been extensively investigated by many previous studies. These studies proved that lead is a potent toxin that affects nervous system, especially children’s brain. However, most of these studies focused on the negative effects of lead on the differentiated or mature cell types in the brains instead of NSCs. The aim of this study was to reveal the effects of Pb2+ on viability, proliferation and differentiation of NSCs derived from the hippocampus of newborn rats aged 7 days and adult rats aged 90 days in vitro. NSCs harvested from the rat hippocampus were cultured in proliferation medium. After 6-8 days, free-floating neurospheres formed. The neurospheres were dissociated and plated onto poly-L-lysine coated 96-well plate and coverslips. Some dissociated cells were characterized by being stained with anti-nestin to show the presence of NSCs. This project was divided into three parts. In the first part, the Passage 2 (P2) cells plated onto 96-well plate were cultured in the proliferation medium with different concentrations of lead acetate (0-200μM) for 48 hours, followed by 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to detect the effects of Pb2+ on the cell viability. In the second part, P2-NSCs plated onto coverslips in wells were cultured in the proliferation medium with different concentrations of lead acetate (0-200μM). Then, 10 μM bromodeoxyuridine (BrdU) was added into the culture medium for additional 24 hours, followed by immunocytochemistry staining with anti-BrdU. In the last part, the dissociated P2-NSCs plated onto coverslips were allowed to grow in the differentiation medium of neurons, astrocytes or oligodendrocytes with different concentrations of lead acetate (0-200μM). After 6 days, immunocytochemistry staining with anti-microtubule-associated protein 2 (anti-MAP2), anti-glial fibrillary acidic protein (anti-GFAP) or anti-RIP was used to detect the differentiation commitment of affected NSCs. Low level of Pb2+ (1-10μM) had no effect on the viability of adult hippocampal neural stem cells (hNSCs). However, Pb2+ exposure at the concentration of 10μM could lead to significant cell death of newborn hNSCs. High level of Pb2+ (50-200μM) caused significant cell death of both newborn and adult hNSCs. Newborn hNSCs were sensitive to Pb2+ toxicity in proliferation assay. Even a low concentration (1μM) of lead could lead to significant inhibition of cell proliferation. High level of Pb2+ (50-200μM) suppressed proliferation of both newborn and adult hNSCs significantly. Moderate to high levels of Pb2+ exposure (50-200μM) significant decreased the percentage of mature neurons cultured from both newborn and adult hNSCs. Furthermore, 10μM or more Pb2+could significantly inhibited the oligodendrocyte differentiation of both newborn and adult hNSCs. However, Pb2+ could also stimulate the astrocyte differentiation of hNSCs. Lead concentrations higher than 10μM and 50μM could respectively lead to a significant increase in the percentage of mature astrocytes differentiated from newborn and adult hNSCs. The data showed that Pb2+ inhibited not only the viability and proliferation of rat hNSCs but also the neuronal and oligodendrocyte differentiation in vitro; moreover activated astrocyte differentiation of the hNSCs of both newborn and adult rats were observed with high concentration of Pb2+ in vitro. Also, it was revealed that the hNSCs of newborn rats were more sensitive than those from adult rats to Pb2+ cytoxicity. / published_or_final_version / Anatomy / Master / Master of Medical Sciences

published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

Neural stem cells (NSCs) exist in both embryonic and adult neural tissues and are characterized by their self-renewal capacity and multipotency that contribute to the generation of three major cell types in the vertebrate central nervous system (CNS):neurons, oligodendrocytes and astrocytes. The tremendous therapeutic potential of NSCs to treat the neurodegenerative diseases and repair brain injuries has provoked intensive study in the molecular regulation of their induction, maintenance and differentiation. Previous study reported that Sox9, a member of high-mobility-group(HMG) containing SoxE transcription factors family, plays important roles in regulating the formation and maintenance of NSCs in both mouse and chick CNS, as well as the cell fate switch between neuronal and glial. Whether it plays similar roles in human NSCs (hNSCs)is still unknown. My RT-qPCR analysis showed that SOX9is expressed at a basal level in human embryonic stem cells (hESCs) and up-regulated upon commitment into neural lineage and maintained at a high level in hESCs-derived hNSCs. I therefore hypothesized that SOX9 might also be involved in the induction, maintenance and differentiation of hNSCs. To test this, two stable hESC lines(HES2)were generated with each constitutively expressing short hairpin RNA (shRNA) against SOX9andGL2 Luciferase (Luc, as control) respectively. Upon neural induction, SOX9-knock-down(KD) hESCs were able to commit neural lineage and differentiate into NSCs/neurospheres (NSPs), however, these NSCs exhibited reduced multipotency and glial marker (GALC, CD44) expressions but enhanced self-renewal compared to the shLuc NSCs. Hence, SOX9 is required for both the induction and maintenance of multipotent hNSCs. Strikingly, extensive TUJ1+ neurites and advance groupings of these neurites into bundles were observed in SOX9-KD NSPs after three days and seven days neuronal differentiation respectively, suggesting premature neurogenesis as a result of SOX9 ablation. In addition, RT-qPCR analysis revealed down-regulated expression of NSC marker HES1but induced proneural basic helix-loop-helix transcription factor MASH1in shSOX9-1208 NSCs. The inhibitory role of HES1 on the expression and functions of MASH1 has been reported to be essential for the timely generation of neurons. Hence, ablation of SOX9 is likely to relieve the inhibition on MASH1activity via down-regulated HES1expression and leads to early neuronal differentiation. Expression of the potent neurite blocker NG2 was also found to be reduced in SOX9-KD NSCs which may explain the extensive neurite network observed. Altogether, similar to previous studies in mouse NSCs, SOX9 is also required for the induction and maintenance of hNSCs. However, this study further reveals a putative novel role of SOX9 in preventing premature neuronal differentiation by regulating the expressions of HES1 to counteract MASH1 function and NG2 to control neurite outgrowth. / published_or_final_version / Biochemistry / Master / Master of Philosophy