Browsing by Subject "human induced pluripotent stem cells"

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    A functional endocannabinoid system in human induced pluripotent stem cell-derived cortical cultures
    (2022-04) Asher, Melissa
    The endocannabinoid system is an increasingly popular therapeutic target in many neurological conditions, due in large part to its ability to protect neurons from damage caused by hyperactivity and excitotoxicity. Despite recent interest in cannabinoid-based treatments, the unavailability of human brain tissue and species differences between humans and animal models present obstacles to drug development. Human induced pluripotent-derived stem cells (hiPSCs), which can be obtained less invasively from skin samples and then reprogrammed into neurons and glia, are one possible solution to this problem. However, it is not clear whether hiPSC-derived neurons actually have a working endocannabinoid system to study. In this thesis I characterize the endocannabinoid system in a commercially available line of hiPSC-derived cortical neuron/astrocyte cultures using calcium imaging and a fluorescent cannabinoid indicator expressed in live neurons. hiPSC-derived cultures produced and metabolized endocannabinoids in addition to responding to exogenously applied cannabinoids, indicating that they do indeed possess a fully functional endocannabinoid system. I also show that endocannabinoid synthesis evoked by a muscarinic receptor agonist in hiPSC-derived cortical cultures is not calcium-dependent, and that an inhibitor of endocannabinoid metabolism produces less receptor desensitization than a cannabinoid receptor agonist with prolonged exposure. These studies demonstrate that hiPSC-derived neuron/astrocyte cultures are a powerful new tool for investigating open questions about the regulation of the human endocannabinoid system.
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    Integrative approaches to understanding the blood-brain barrier in the context of Alzheimer’s disease
    (2024-09) Eberts, Paulina
    The blood-brain barrier (BBB) is a highly selective barrier that is crucial for maintaining brain homeostasis. Disruption of the BBB can have deleterious effects, and it is believed that certain types of BBB damage are central to the etiology of Alzheimer's disease (AD). Given its role in disease onset, disrupted BBB function could be an attractive target for potential early intervention in AD. However, more needs to be understood about its functions in relation to AD to best inform these approaches. This includes understanding the relationship between the BBB and the multitude of factors thought to contribute to AD development, including genetic and nongenetic risk factors. To facilitate this, improved models and strategies are required to identify mechanistic links between risk factors and early features of AD. Animal models have been indispensable in AD research, providing critical insights into disease mechanisms. However, they are limited in their ability to elucidate the specific interactions between hallmark features of AD and the BBB endothelium, partly due to critical species differences. Human in vitro models of the BBB offer a complementary approach, allowing for a highly controlled, tunable environment for the examination of complex, multifactorial interactions with greater throughput. This makes them well-suited for a systems approach, which is particularly beneficial for understanding complex diseases like AD. However, these models also have limitations, such as their lack of maturity, which decreases their relevance to a disease that primarily affects the aged adult population. Despite this, there are considerable opportunities to expand the types of insights that can be gained from them. The purpose of this dissertation is to enhance understanding of BBB functions as they relate to the earliest stages of AD through efforts to improve in vitro models, apply these models, and expand the model toolkit. In Chapter 1, the fundamentals of AD are reviewed and the role of the BBB in this context is presented. This includes a summary of models and approaches typically used to examine the BBB in relation to AD. Chapter 2 describes methods for improving models of the BBB comprised of brain microvascular endothelial-like cells (iBMECs) derived from human induced pluripotent stem cells (hiPSCs) to better represent the adult BBB. This work finds that extending the culture of iBMEC models of the BBB induces quiescence and improves structural organization in the barriers, indicative of a more mature, rested phenotype. Chapter 3 presents the application of in vitro models of the BBB to map out and characterize amyloid-beta binders expressed by the brain endothelium. This work identifies HspB1 as a key amyloid-beta binder expressed by the brain endothelium. Further examination of HspB1 links its behavior to features of genetic and non-genetic risk for AD, namely ApoE isoform and oxidative stress. Chapter 4 discusses the extension of a proximity labeling technology, TurboID, for use in identifying protein-protein interactions at the BBB surface. This includes recombinant expression of TurboID constructs for extracellular use. Design principles determined in AlphaFold for the robust design of constructs implicate linker selection in ensuring proper construct binding activity. Chapter 5 discusses key conclusions from each of these efforts, as well as future directions.