Investigating the existence of neural stem cells in the adult mouse cerebellum and third ventricle

In mammals, adult neurogenesis in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus produces neurons contribute to learning and memory functions. However, more recent evidence suggests that neurogenesis may also happen in other regions of the brain such as striatum, spinal cord, and hypothalamus (Reviewed in (Riddle, 2007)). It is important to determine if neurogenesis also occurs in non-neurogenic regions of the adult brain such as CB and 3V. This would be of importance for potential future therapeutic applications for brain repair, and also to help understand the fundamental function of the different regions of the adult brain. This thesis tested the hypothesis that neural stem cells (NSCs) are present in the mature cerebellum (CB) and the lining of the third ventricle (3V). Based on this hypothesis, one of the major goals of this thesis was to isolate and characterise NSCs isolated from CB and 3V of adult mouse and weather they could generate neuronal and glial cells in vitro. Immunohistochemical analysis for NSC-associated markers revealed that the mature CB in mouse, chick, and primates contains a population exhibiting NSCs characteristics. Results showed that this population was the Bergmann glial located in the Purkinje cell layer (PCL) of the cerebellar cortex, which express Sox1, Sox2, Sox9, BLBP, and GFAP in a similar pattern. Some of these markers are common for neural stem/progenitor cells or radial glial cells in other brain regions. Observations in the 3V revealed that tanycytes lining the ependymal layer also express NSC and astrocytic markers. Moreover, cells were isolated from the CB and 3V of adult GFP+/-Sox1 mice and tested their ability to form neurospheres, their response to EGF and FGF-2, and their differentiation into neurons, astrocytes, and oligodendrocytes. CB and 3V-isolated cells were found to grow in culture, expand, and differentiate into neuronal and glial phenotypes. It was also observed that CB-derived NSCs could survive and differentiate into neuronal and glial lineages after long term removal of either EGF or FGF-2, although cultures were optimal in the presence of both mitogens. We also observed that cells cultured in either EGF or FGF-2 for 3 weeks had different effects on both CB and LV cells in terms of cell fate specification toward neuronal and glial lineages. This finding suggests the heterogeneity of NSCs population in the adult brain. The identification and mapping of the different NSC populations present in the adult brain offers some important opportunities for regenerative medicine approaches. In order to better characterise the cerebellar population of cells identified above, adult Bergmann glial was observed in a mouse model of cerebellar damage caused by the loss of PCs in pcd5J mutant mice. Calbindin immunostaining at different time-points showed that PC degeneration was visible at P21, then progressed rapidly and became considerable at P26 (nearly 70-80% loss), and by P100 all PCs were lost. Immunohistochemical analysis on sections of CB from pcd and wild type counterparts revealed an increase in Bergmann glia cells at P100 as well as the upregulation of GFAP expression. GFAP+ BG exhibited thick disorganised processes in the molecular layer (ML) at P100 in the mutant mice, with some cell bodies mispositioned in the ML, and significant shrinkage of both ML and internal granular layer (IGL). The increase in the number of Sox1+, Sox2+, and Sox9+ BG in 3 month old mutant mice was not visible at earlier time points analysed. These findings indicate that the PCs loss in pcd5J mice precede and possibly trigger the increase in the Sox1+, Sox2+ and Sox9+ cell population in the CB. Our results also showed that no proliferation activity was observed in the pcd mouse CB at revealed by Ki67 staining, suggesting that the CB microenvironment might not be permissive for neurogenesis even after PCs loss. In vitro isolation of NSCs the CB of P21 pcd mice was carried out, and although cultures appear slower to establish than wild type controls these cells did form neurospheres and express NSC markers. Further characterisation of CB-derived NSCs from pcd mice and their growth and differentiation potential will help better understand the dynamics and possible therapeutic targets for neurodegenerative disorders affecting the CB. The characterisation of CB and 3V derived NSCs from adult mouse CB has provided important information regarding their differences with NSCs derived from neurogenic region in the brain, the lateral ventricle (LV)

Investigating the existence of neural stem cells in the adult mouse cerebellum and third ventricle

In mammals, adult neurogenesis in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus produces neurons contribute to learning and memory functions. However, more recent evidence suggests that neurogenesis may also happen in other regions of the brain such as striatum, spinal cord, and hypothalamus (Reviewed in (Riddle, 2007)). It is important to determine if neurogenesis also occurs in non-neurogenic regions of the adult brain such as CB and 3V. This would be of importance for potential future therapeutic applications for brain repair, and also to help understand the fundamental function of the different regions of the adult brain. This thesis tested the hypothesis that neural stem cells (NSCs) are present in the mature cerebellum (CB) and the lining of the third ventricle (3V). Based on this hypothesis, one of the major goals of this thesis was to isolate and characterise NSCs isolated from CB and 3V of adult mouse and weather they could generate neuronal and glial cells in vitro. Immunohistochemical analysis for NSC-associated markers revealed that the mature CB in mouse, chick, and primates contains a population exhibiting NSCs characteristics. Results showed that this population was the Bergmann glial located in the Purkinje cell layer (PCL) of the cerebellar cortex, which express Sox1, Sox2, Sox9, BLBP, and GFAP in a similar pattern. Some of these markers are common for neural stem/progenitor cells or radial glial cells in other brain regions. Observations in the 3V revealed that tanycytes lining the ependymal layer also express NSC and astrocytic markers. Moreover, cells were isolated from the CB and 3V of adult GFP+/-Sox1 mice and tested their ability to form neurospheres, their response to EGF and FGF-2, and their differentiation into neurons, astrocytes, and oligodendrocytes. CB and 3V-isolated cells were found to grow in culture, expand, and differentiate into neuronal and glial phenotypes. It was also observed that CB-derived NSCs could survive and differentiate into neuronal and glial lineages after long term removal of either EGF or FGF-2, although cultures were optimal in the presence of both mitogens. We also observed that cells cultured in either EGF or FGF-2 for 3 weeks had different effects on both CB and LV cells in terms of cell fate specification toward neuronal and glial lineages. This finding suggests the heterogeneity of NSCs population in the adult brain. The identification and mapping of the different NSC populations present in the adult brain offers some important opportunities for regenerative medicine approaches. In order to better characterise the cerebellar population of cells identified above, adult Bergmann glial was observed in a mouse model of cerebellar damage caused by the loss of PCs in pcd5J mutant mice. Calbindin immunostaining at different time-points showed that PC degeneration was visible at P21, then progressed rapidly and became considerable at P26 (nearly 70-80% loss), and by P100 all PCs were lost. Immunohistochemical analysis on sections of CB from pcd and wild type counterparts revealed an increase in Bergmann glia cells at P100 as well as the upregulation of GFAP expression. GFAP+ BG exhibited thick disorganised processes in the molecular layer (ML) at P100 in the mutant mice, with some cell bodies mispositioned in the ML, and significant shrinkage of both ML and internal granular layer (IGL). The increase in the number of Sox1+, Sox2+, and Sox9+ BG in 3 month old mutant mice was not visible at earlier time points analysed. These findings indicate that the PCs loss in pcd5J mice precede and possibly trigger the increase in the Sox1+, Sox2+ and Sox9+ cell population in the CB. Our results also showed that no proliferation activity was observed in the pcd mouse CB at revealed by Ki67 staining, suggesting that the CB microenvironment might not be permissive for neurogenesis even after PCs loss. In vitro isolation of NSCs the CB of P21 pcd mice was carried out, and although cultures appear slower to establish than wild type controls these cells did form neurospheres and express NSC markers. Further characterisation of CB-derived NSCs from pcd mice and their growth and differentiation potential will help better understand the dynamics and possible therapeutic targets for neurodegenerative disorders affecting the CB. The characterisation of CB and 3V derived NSCs from adult mouse CB has provided important information regarding their differences with NSCs derived from neurogenic region in the brain, the lateral ventricle (LV)

Magnetically Assisted Control of Stem Cells Applied in 2D, 3D and In Situ Models of Cell Migration

The success of cell therapy approaches is greatly dependent on the ability to precisely deliver and monitor transplanted stem cell grafts at treated sites. Iron oxide particles, traditionally used in vivo for magnetic resonance imaging (MRI), have been shown to also represent a safe and efficient in vitro labelling agent for mesenchymal stem cells (MSCs). Here, stem cells were labelled with magnetic particles, and their resulting response to magnetic forces was studied using 2D and 3D models. Labelled cells exhibited magnetic responsiveness, which promoted localised retention and patterned cell seeding when exposed to magnet arrangements in vitro. Directed migration was observed in 2D culture when adherent cells were exposed to a magnetic field, and also when cells were seeded into a 3D gel. Finally, a model of cell injection into the rodent leg was used to test the enhanced localised retention of labelled stem cells when applying magnetic forces, using whole body imaging to confirm the potential use of magnetic particles in strategies seeking to better control cell distribution for in vivo cell delivery