Engineering Tools to Study and Control Signaling in Neural Stem Cells

Adult neurogenesis, the process by which new neurons develop in the adult mammalian central nervous system, was thought to be nonexistent by accepted scientific dogma until the discovery of adult neural stem cells (NSCs) in the 1990s. NSCs have been found in two regions of the adult brain: the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus, and have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes. Importantly, hippocampal NSCs play key roles in learning and memory, and have been implicated in a number of pathologies, including Alzheimer’s disease. NSCs reside in complex niches that provide the physical, chemical, and biological signals regulating stem cell maintenance and differentiation. A thorough understanding of NSC biology and niche signals can provide both insight into the mechanisms of adult neurogenesis, and inform stem cell based therapeutics to treat neurological injury and disease. Since the discovery of NSCs, a body of work has emerged characterizing the wide array of signals and intracellular pathways that mediate NSC behavior. Some of these findings, however, point to complex signaling mechanisms, the further study of which requires techniques outside the standard biological “toolbox”. The goal of this dissertation, therefore, was to engineer novel tools to enable the study and control of complex signaling systems in NSCs, and their application towards novel biological discoveries.The work presented here investigates two aspects of NSC biology: heterogeneity and cell-cell signaling. Stem cells are inherently heterogeneous; NSCs give rise to diverse progeny, including neurons and astrocytes, and in vitro, NSCs can differentially respond to the same set of cues. To probe this heterogeneity, a single cell Western blotting (scWestern) platform was developed. scWesterns enabled the interrogation of the proteome of thousands of single cells in about four hours, and multiple