Quiescent neural stem cells support continuous, lifelong neurogenesis in specific regions of the adult mammalian brain. The largest adult neurogenic region is the ventricular-subventricular zone (V-SVZ), which lines the entire lateral wall of the lateral ventricles. Quiescent neural stem cells (qNSCs) enter the cell cycle (activate) and give rise to new neurons during homeostasis and regeneration, suggesting they can potentially be harnessed for regenerating the brain after neurodegenerative disease, stroke, and injury. Defining the signals that regulate NSC quiescence and activation is essential to unlock their potential for regenerative medicine. NSCs residing in specific regions of the V-SVZ give rise to distinct subtypes of olfactory bulb interneurons. It is unknown whether quiescence-regulating signals map onto the regional heterogeneity of NSCs, and might thereby underlie the production of distinct interneuron subtypes. A major limitation to our understanding of the regulation of NSC quiescence has been the lack of specific markers to identify qNSCs, and prospectively purify them from their in vivo niche. Using a novel fluorescence-activated cell sorting (FACS) strategy that allows the purification of qNSCs from the adult mouse V-SVZ niche for the first time, I performed in vitro screens for quiescence-regulating signals. Unexpectedly, neurotransmitters emerged as the main class of qNSC-activating signals, including dopamine, GABA, serotonin, acetylcholine, and opioids. Local and long-range neurons that use these neurotransmitters innervate the V-SVZ in unique regional patterns, suggesting these signals map onto the regional heterogeneity of NSCs. Consistent with this hypothesis, infusions of cholinergic agonist and antagonists into the lateral ventricle resulted in regional changes in NSC proliferation. Moreover, cholinergic antagonists blocked the activation of qNSCs during regeneration, providing evidence that neurotransmitter signaling activates qNSCs in vivo. I then showed that hypothalamic Pomc-expressing neurons innervate the anterior-ventral V-SVZ and promote the activation of Nkx2.1+ qNSCs. Ablation of Pomc+ neurons resulted in decreased proliferation of NSCs in the anterior-ventral, but not anterior-dorsal, V-SVZ. Moreover, both the activity of Pomc+ neurons, and the proliferation of Nkx2.1+ NSCs in the anterior-ventral V-SVZ decreased in fasted animals, suggesting that hunger and satiety states regulation the generation of a single olfactory bulb interneuron subtype. Indeed, ablation of Pomc+ neurons resulted in a loss of the subtype of olfactory bulb interneuron that is generated by Nkx2.1+ NSCs. Together, my findings suggest that both local and long-range neurons regionally innervate the V-SVZ and mediate neural stem cell activation from the quiescent state.

Mammalian neural stem cell (NSC) lines provide a useful experimental model for basic and applied research across stem cell and developmental biology, regenerative medicine and neuroscience. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with functional genetic analyses. However, targeted genetic manipulations have not been reported for mammalian NSC lines. Here, we deploy the CRISPR/Cas9 technology and demonstrate a variety of diverse targeted genetic modifications in both mouse and human NSC lines such as: targeted transgene insertion at safe harbour loci; biallelic knockout of neurodevelopmental genes; knock-in of epitope tags and fluorescent reporters; and engineering of glioma driver mutations at endogenous proto-oncogenes. Leveraging these new optimised methods, we explored gene editing to model the earliest stages of paediatric gliomagenesis in primary human NSCs. Our data indicate that oncogenic mutations in histone H3.3 play a role in NSC transformation and may operate through suppression of replication induced senescence. By comparing cellular responses of NSC cultures from different compartments of the developing brain, we also identify differences in susceptibility to distinct H3.3 mutations that mirror the disease etiology. Altogether, our findings indicate that CRISPR/Cas9-assisted genome editing in NSC lines is a versatile tool to explore gene function in CNS development and cancer biology. Our project resulted in the creation of valuable human cellular models of paediatric gliomagenesis, which will allow us to further our understanding of the disease and carry out cell based drug discovery projects.

Remarkable progress has been made in our ability to design and produce synthetic DNA binding domains (TALE or Cas9-based), which can be further functionalized into synthetic transcription factors (sTFs). This technology is revolutionizing our ability to modulate expression of endogenous mammalian genes. Forced expression of cDNAs encoding transcription factors (TFs) is widely used to drive lineage conversions. However, this process is often inefficient and unreliable. Multiplex delivery of sTFs pool to activate endogenous master regulators and extinguish the expression profile of the host cell type could be a potential solution to this problem. We have developed a novel, simple TALE assembly method that enabled us to produce and screen large numbers of TAL effectors and compare their activity to dCas9-based TFs. During this process, we constructed many new functionally validated sTFs. Our ultimate goal is to test whether combining synthetic transcriptional activators and repressors can efficiently reprogram fibroblasts to NS cells or alternatively ‘program’ NS cell differentiation to neurons. We performed analyses of the transcriptome and chromatin accessibility of both fibroblasts and neural stem cells to unravel their core TF networks and their epigenetic state. This will allow us in the future the targeted design of sTFs and synthetic chromatin modifiers for specifically changing cell identity.

Au cours de ce travail, nous avons cherché de préciser le rôle de cette famille de facteur de croissance dans le choix dun destin cellulaire au cours du développement, tout en gardant à lesprit leur éventuel potentiel dans le cadre dune thérapie cellulaire chez ladulte. Nous avons donc tenté de répondre aux questions suivantes : 1) est-il possible dinfluencer le choix dun destin cellulaire particulier dans les cellules souches nerveuses sous leffet des Neurégulines ; 2) si oui, quel(s) serait (seraient) le(s) mécanisme(s) moléculaire(s) éventuellement recrutable ou applicable en terme de régénération. Le chapitre II est consacré à létude des Neurégulines-1 et leurs effets sur la prolifération et la différenciation des cellules souches nerveuses in vitro. Dans ce chapitre, nous décrivons un mécanisme moléculaire original responsable de la modulation de la différenciation par les Neurégulines-1 et qui établit un line direct entre les influences intrinsèques et extrinsèques telles que nous les avons rappelées en préambule du point 2 de cette introduction (Edlund and Jessell, 1999). La plupart des résultats de ce chapitre font lobjet dun article actuellement sous presse (appendice 2). Dans le chapitre III, nous décrivons les résultats préliminaires obtenus dans létude du rôle du rôle de Nrg-2 dans ces mécanismes. Cest sur la base de la similitude structurelle et topologique des isoformes codées par les deux gènes que nous avons entrepris cette dernière partie de notre travail.

In embryonic stem cells (ESCs) neural differentiation is elicited in the absence of extrinsic signaling in minimal conditions. This ‘default mechanism’ in ESCs produces neural stem cells termed primitive neural stem cells, which can subsequently yield FGF2-dependent definitive neural stem cells (dNSCs). We hypothesized that dNSCs have properties similar to neural stem/progenitor cells derived from the adult brain (aNPCs). The neural differentiation profile of the cell-types was characterized in vitro and in vivo following transplantation into the Shiverer mouse. The dNSCs produced a differentiation profile similar to that of aNPCs and both cell-types produced oligodendrocytes. This is the first demonstration of the in vivo differentiation of neural stem cells, derived from ESCs through the default mechanism, into the oligodendrocyte lineage. We conclude that dNSCs are a similar cell population to aNPCs. The default mechanism is a promising approach to generate neural stem cells and their progeny from pluripotent cell populations.

In embryonic stem cells (ESCs) neural differentiation is elicited in the absence of extrinsic signaling in minimal conditions. This ‘default mechanism’ in ESCs produces neural stem cells termed primitive neural stem cells, which can subsequently yield FGF2-dependent definitive neural stem cells (dNSCs). We hypothesized that dNSCs have properties similar to neural stem/progenitor cells derived from the adult brain (aNPCs). The neural differentiation profile of the cell-types was characterized in vitro and in vivo following transplantation into the Shiverer mouse. The dNSCs produced a differentiation profile similar to that of aNPCs and both cell-types produced oligodendrocytes. This is the first demonstration of the in vivo differentiation of neural stem cells, derived from ESCs through the default mechanism, into the oligodendrocyte lineage. We conclude that dNSCs are a similar cell population to aNPCs. The default mechanism is a promising approach to generate neural stem cells and their progeny from pluripotent cell populations.

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Biomedical Engineering." Includes bibliographical references (p. 53-55).

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain brings promise for the treatment of neurological diseases and has yielded new insight into brain cancer. The complete repertoire of signaling pathways that governs these cells however remains largely uncharacterized. This thesis describes how chemical genetic approaches can be used to probe and better define the operational circuitry of the NSC. I describe the development of a small molecule chemical genetic screen of NSCs that uncovered an unappreciated precursor role of a number of neurotransmitter pathways commonly thought to operate primarily in the mature central nervous system (CNS). Given the similarities between stem cells and cancer, I then translated this knowledge to demonstrate that these neurotransmitter regulatory effects are also conserved within cultures of cancer stem cells. I then provide experimental and epidemiologically support for this hypothesis and suggest that neurotransmitter signals may also regulate the expansion of precursor cells that drive tumor growth in the brain. Specifically, I first evaluate the effects of neurochemicals in mouse models of brain tumors. I then outline a retrospective meta-analysis of brain tumor incidence rates in psychiatric patients presumed to be chronically taking neuromodulators similar to those identified in the initial screen. Lastly, by further exploring the phenotype and function of neurotransmitter pathways in purified populations of human NSCs, I determined that neurotransmitter pathway gene expression exists in a functionally heterogeneous phase-varying state that restricts the responsiveness of these populations to various stimuli. Taken together, this research provides novel insights into the phenotypic and functional landscape of neurotransmitter pathways in both normal and cancer-derived NSCs. In additional to a better fundamental understanding of NSC biology, these results suggest how clinically approved neuromodulators can be used to remodel the mature CNS and find application in the treatment of brain cancer.

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain brings promise for the treatment of neurological diseases and has yielded new insight into brain cancer. The complete repertoire of signaling pathways that governs these cells however remains largely uncharacterized. This thesis describes how chemical genetic approaches can be used to probe and better define the operational circuitry of the NSC. I describe the development of a small molecule chemical genetic screen of NSCs that uncovered an unappreciated precursor role of a number of neurotransmitter pathways commonly thought to operate primarily in the mature central nervous system (CNS). Given the similarities between stem cells and cancer, I then translated this knowledge to demonstrate that these neurotransmitter regulatory effects are also conserved within cultures of cancer stem cells. I then provide experimental and epidemiologically support for this hypothesis and suggest that neurotransmitter signals may also regulate the expansion of precursor cells that drive tumor growth in the brain. Specifically, I first evaluate the effects of neurochemicals in mouse models of brain tumors. I then outline a retrospective meta-analysis of brain tumor incidence rates in psychiatric patients presumed to be chronically taking neuromodulators similar to those identified in the initial screen. Lastly, by further exploring the phenotype and function of neurotransmitter pathways in purified populations of human NSCs, I determined that neurotransmitter pathway gene expression exists in a functionally heterogeneous phase-varying state that restricts the responsiveness of these populations to various stimuli. Taken together, this research provides novel insights into the phenotypic and functional landscape of neurotransmitter pathways in both normal and cancer-derived NSCs. In additional to a better fundamental understanding of NSC biology, these results suggest how clinically approved neuromodulators can be used to remodel the mature CNS and find application in the treatment of brain cancer.

Thesis (Ph.D.)--Ohio University, March, 2009. / Release of full electronic text on OhioLINK has been delayed until April 1, 2011. Includes bibliographical references (leaves 99-108)