Browsing by Subject "Neural Stem Cells"
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Item Alterations in Neural Stem Cell Fate Following Focal Ischemia(2014-02-04) Nguyen, Derek; Vemireddy, Vamsidhara; Mashimo, Tomoyuki; Battiste, James; Maher, Elizabeth; Bachoo, RobertINTRODUCTION: The purpose of this experiment is to determine the differentiation identity of the neural stem cells (NSC) in the subventricular zone (SVZ) of adult mouse brain after a middle cerebral artery occlusion (MCAO). Injury to the brain causes a large number of changes including inflammation and apoptosis, but the reaction of NSC's has been more difficult to characterize because of the transient nature of their response. Previously, adult neural stem cells (NSC) in the SVZ have been observed to differentiate predominantly into cells with neuronal characteristics. This theory is questioned via a tamoxifen-inducible cre-recombinase (Cre-ERT2) expression mouse model system. METHOD: The Cre-ERT2 expression mouse model system is driven by the Cystatin-C promoter to label NSC's in a time specific manner and track their cell fate after MCAO. After the ischemia, these brain sections were stained with different immunohistochemicals at three separate time points. One set was co-labeled with GFAP, an astrocyte marker, and BrdU, a proliferation marker. Another set was co-labeled with DCX, a neuronal marker, and BrdU. This was used to differentiate between latent NSCs and proliferating NSCs by comparing the ipsilateral side (ischemic) with the contralateral side (control) of the brain. RESULTS: Compared to the contralateral, the ipsilateral side had a significant increase in GFAP/BrdU positive cells between day 3 and day 7 time points. The cell quantity dropped between day 7 to day 14 time points. Compared to the contralateral, the ipsilateral side had a decrease in DCX/BrdU positive cells between day 3 and day 7 time points. The cell quantity significantly increased between day 7 to day 14 time points, and the quantity at day 14 was about twice to that of the day 3 time point. DISCUSSION: This data demonstrated that after the MCAO, the stem cells are not just undergoing neurogenesis, but are for certain period of time, also differentiating into astrocytes that are migrating towards the site of injury. This phenomenon is only witnessed in the NSCs towards the day 7 time point. Afterwards and leading up to day 14, the NSCs seem to be changing their cell fate programming from the astrocyte pathway back to the intended neuronal pathway. Thus, the staining results verify that after an ischemia, NSCs within the SVZ regions of the brain undergo a constant change of programmed cell fate, alternating between immature neurons and astrocytes implicating future aims for "programmed" neurogenesis in the development of therapeutic strategies for the treatment of brain damage and disease.Item Neural Stem Cells in Brain Tumor Development(2009-09-04) Llaguno, Sheila R. Alcantara; Parada, Luis F.Malignant astrocytomas are highly invasive and incurable brain tumors. Mouse models that genetically resemble the human disease are valuable tools in understanding the pathogenesis of these malignancies. We previously reported mouse models based on conditional inactivation of the human astrocytoma-relevant tumor suppressors Nf1, p53 and Pten. Through somatic loss of heterozygosity, these mice develop varying grades of astrocytic malignancy with 100% penetrance. Studies on our tumor suppressor mouse models indicated a central role for neural stem cells and stem cell-like cancer cells in malignant astrocytoma formation. Using stereotactic viral cre-mediated approach, we demonstrate that targeting of tumor suppressor inactivating mutations in the subventricular zone (SVZ) where neural stem and progenitor cells reside is both necessary and sufficient to induce astrocytoma formation. We also show evidence of spontaneous differentiation and infiltration of these cancer-initiating cells in situ during tumor development. These studies have so far shown that neural stem cells or its progeny can give rise to astrocytomas. Neural stem cells, which have unlimited self-renewal potential, produce transit amplifying cells, or progenitor cells, which undergo limited mitoses before differentiating into more mature cell types. By genetically targeting transit amplifying cells using the Ascl1-creERT2 transgenic mouse, we show that tumor suppressor inactivation in the progenitor compartment alone induces malignant astrocytoma formation. Defects in proliferation, differentiation, and migration are likewise found several months prior to advanced disease. This establishes both neural stem and progenitor cells as cells of origin of malignant astrocytomas in our tumor suppressor mouse models. In another study, we isolated and characterized a population of stem cell-like cancer cells from murine astrocytomas that are enriched for tumor cells compared to primary tumor tissue, exhibit aberrant stem cell properties, and are tumorigenic in vivo. We demonstrate resistance to a known chemotherapeutic agent and the migratory capacity of these cells. We also investigated the mechanisms involved in astrocytoma progression and maintenance by gene expression analysis. Genomic profiling of tumor-derived neurosphere-forming cells from conditional astrocytoma mouse models show prominent dysregulation of genes involved in neurodevelopmental processes and transcriptional regulation, particularly the hox transcription factors, in high-grade astrocytomas. Taken together, we have demonstrated that neural stem and progenitor cells are the origins of malignant astrocytoma in tumor suppressor mouse models. We have established a system by which molecular mechanisms of tumor development can be further investigated and performed genomic profiling of tumor-derived neurosphere-forming cells, suggesting a possible role for homeobox transcription factors in malignant astrocytoma formation. These mouse models thus represent powerful tools in understanding various aspects of cancer development that otherwise cannot be explored in humans. Further studies will provide a better understanding of the biology of these tumors and will hopefully pave the way for more effective therapeutic approaches for these devastating diseases.Item Novel Astrocyte-Specific Transgenic Mice Identify Distinct Populations of Transient Amplifying Progenitor Cells And Long-Lived Neural Stem Cells In the Subgranular Zone of the Adult Mouse Brain(2011-02-01) Boies, Lori Nicole Loomis; Bachoo, RobertA lack of previous interest in the astrocyte biology field has resulted in a meager number of astrocyte specific molecular markers and practical protocols to study their biology and function. The most common and accepted astrocyte marker is glial fibrillary acidic protein (GFAP), but in normal, healthy adult brains, GFAP expression is limited to the neurogenic regions, white matter tracks, and pia. This limited expression is not a true representation of astrocyte distribution patterns and does not account for astrocyte heterogeneity. Through a careful screen, cystatin C (CstC) and phospholipase A2 Group VII (PLA2G7) were identified as astrocyte specific molecular markers. To study the role of these genes in astrocyte biology, inducible CreERT2 mice were generated. Using the CstC-CreERT2 and PLA2G7-CreERT2 mouse lines, in concert with the Rosa26YFP reporter line, cell fate tracking experiments were performed in both embryonic and adult time points in tandem with extensive immunohistochemistry. Both astrocyte-specific CreERT2 lines demonstrated limited expression in embryos as early as embryonic day 12.5 and extensive expression throughout the entire central nervous system in all adult time points analyzed. In adult animals, both CstC-CreERT2 and PLA2G7-CreERT2 Rosa26YFP positive cells had strong expression in both neurogenic niches. Using careful quantification and immunohistochemistry colocalization, the dynamics of these transgenic lines were meticulously researched in the subgranular zone (SGZ) of the dentate gyrus. The CstC-CreERT2/Rosa26YFP cells represent a transient amplifying compartment in the SGZ while PLA2G7-CreERT2/Rosa26YFP positive cells comprise a long-lived neural stem cell compartment. Discovery of these astrocyte dynamics in the SGZ complements the literature indicating astrocytes are the true neural stem cells and adds knowledge of new viable markers and cell populations to the field. Results of these studies reveal not only new molecular markers for astrocytes in both embryonic and adult scenarios, but also help to identify the underlying heterogeneity of astrocyte populations, as well as adult neural stem cells. Future utilization of these inducible transgenic mouse models could provide the potential to study both astrocyte and neural stem cell pathophysiologies.Item Roles of Ascl1 and Olig2 in the Transcriptional Regulation of Astrocytogenesis(2016-01-19) Combiths, Adam; Vue, Tou Yia; Johnson, Jane E.Ascl1 and Olig2 are transcription factors highly expressed in certain neural progenitor cells, and are known to be involved in neurogenesis and oligodendrogenesis (OL) throughout the CNS; their role in astrocytogenesis (AS) is less well explored. Recent evidence shows that Ascl1-lineage AS clones in the spinal cord (SC) are spatially restricted to either gray matter (GM) or white matter (WM), but not both, and that Olig2 may be necessary for WM astrocytogenesis in the brain. We consider the following questions: (1) Do Ascl1+ progenitors give rise to astrocytes in the brain? (2) Do astrocyte clones, in general, display the GM/WM spatial restriction seen in Ascl1-lineage astrocytes? (3a) Is Olig2 expressed by astrocytes in the SC? (3b) If it is, is this expression required for astrocytogenesis in the SC? To address (1), we used the CreERT2 system under the Ascl1 promoter to label Ascl1+ progenitor cells in the neonatal murine brain (and their progeny) with the tdTom fluorescent reporter. Adult brains were obtained and immunohistochemically (IHC) labeled for factors specific to mature AS, OL and neural lineages; AS, OL, and neurons derived from neonatal Ascl1+ progenitors were observed in every major cortical and subcortical structure, showing that neonatal Ascl1+ progenitors do give rise to AS through the brain. To address (2), we used the CreERT2-Confetti system under the promoter for hGFAP (an astrocyte-specific marker) to give sparse labeling of astrocytes in multiple colors, so that any clone (one clone representing all the progeny of a single AS-progenitor cell) will be far from and visually distinct from other clones. Adult murine SCs were obtained, sectioned, and analyzed by fluorescence microscopy. The location, morphology and clonal identity of every labeled cell was cataloged and used to construct a clonal map of AS distribution in the spinal cord from neonatal development through adulthood, revealing the presence of "mixed" (non-GM/WM-restricted) AS clones - strong evidence for the existence of a GM/WM bipotent AS progenitor cell. To address (3a), we used the CreERT2 system under hGFAP, and IHC labeled for the presence of Olig2. The presence of Olig2+;tdTom+ double positive cells (i.e., astrocytes expressing Olig2) was quantified via fluorescence microscopy. Approximately 50% of astrocytes expressed Olig2. To address (3b), we repeated the above procedure in mice with floxed Olig2 alleles, allowing conditional knockout (CKO) of Olig2 at time of induction. 50% of Olig2-CKO spinal cords showed an almost-complete lack of astrocytes, tentatively indicating a vital role of Olig2 expression in astrocytogenesis in both the GM and the WM.Item Roles of HDACs and MEF2 in Adult Hippocampal Neurogenesis(2014-11-17) Jiang, Yindi; Zhang, Chun-Li; Olson, Eric N.; Johnson, Jane E.; Hsieh, JennyThe maintenance of the resident adult neural stem/progenitor cell (NSPC) pool depends on the precise balance of proliferation, differentiation, and maintenance of the undifferentiated state. Identifying the mechanisms that regulate this balance in adult hippocampal NSPCs can provide insight into basic neurogenesis principles important for tissue homeostasis and preventing tumor formation. Pharmacological inhibition of histone deacetylases (HDACs), a class of histone-modifying enzymes, have promising effects in cancer cells, yet the specific roles of individual HDACs in adult NSPCs are unclear. In this dissertation, I focus on dissecting the roles of two different HDACs in adult hippocampal neurogenesis: the Class I HDAC, HDAC3 and the Class IIa HDAC, HDAC5 as well as the Class IIa HDAC binding partner, myocyte enhancer factor 2 (MEF2). Using conditional knockout (cKO) mice and in vitro cell culture, I show that histone deacetylase 3 (HDAC3) is required for the proliferation of adult NSPCs. Detailed cell cycle analysis of NSPCs from Hdac3 cKO mice reveals a defect in cell cycle progression through G2/M phase, but not S phase. Moreover, HDAC3 controls G2/M phase progression mainly through post-translational stabilization of the G2/M cyclin- dependent kinase-1 (CDK1). These results demonstrate that HDAC3 plays a critical role in NSPC proliferation. HDAC5 is the most abundant Class IIa HDAC in adult dentate gyrus. HDAC5 is only expressed in immature and mature neurons. Using Hdac5 knockout mice and in vitro cell culture, I show that HDAC5 is necessary and sufficient to restrict the neuronal differentiation of NSPCs. However, the detailed mechanisms are yet to be determined. Class IIa HDACs bind to myocyte enhancer factor 2 (MEF2) in the nucleus to repress transcription of pro-neuronal genes. Thus, we also examined the function of Mef2 genes in adult hippocampal neurogenesis. In adult hippocampus, the three most highly expressed MEF2 proteins are MEF2A, 2C, and 2D, which are expressed in immature and mature neurons similar to HDAC5. We have shown that one synthetic small molecule, Isoxazole-9 (Isx-9) could trigger neuronal differentiation robustly in vitro and in vivo. Inducible knockout of all three Mef2 genes specifically in NSPCs and their progeny revealed their critical roles in mediating Isx-9 induced neurogenesis and baseline neurogenesis. In summary, these results demonstrate that HDACs and MEF2 control different stages of adult hippocampal neurogenesis and suggest that strategies aimed at pharmacological modulation of these proteins may be beneficial for tissue regeneration and controlling tumor cell growth in mammalian brain.Item Transcriptional Regulation of Adult Neurogenesis by NRSF/REST and NeuroD1(2011-08-10) Ure, Kerstin Maria; Hsieh, JennyNeurogenesis in the adult brain is a complex and lifelong process that is regulated by multiple pathways and is sensitive to many external stimuli. Two critical regulatory factors in this process are NRSF/REST and NeuroD1. NRSF/REST, a transcriptional repressor that binds a specific NRSE site and recruits corepressors and chromatin remodeling machinery to repress its target genes, is critical for maintenance of the neural stem cell pool and for proper pacing of neuronal differentiation. NeuroD1, a bHLH transcription factor, is necessary for the terminal differentiation, maturation, and survival of newborn neurons. In addition, both factors are necessary for the neurogenic response to both physiological and pathological stimuli, which may induce neurogenesis through different pathways. Thus, NRSF/REST and NeuroD1 are necessary for neurogenesis to occur correctly, to persist throughout the organism’s lifespan, and to respond to external stimuli.