In Vitro Modeling of the Central Nervous System: Towards Optimized Cell-based Therapies


Cell-based therapy is a promising treatment option for central nervous system (CNS) disease and injury, but therapy strategies require optimization to achieve clinical improvement. The primary motivation of this thesis is to gain knowledge of in vitro neuronal differentiation of stem cells and to develop three-dimensional (3D) culture models to investigate stem cell transplantation to the CNS. The first study in this thesis addressed the heterogeneity of mouse embryonic stem cell-derived dopamine neurons. In vitro, the percentage of calbindin+ subtype of DA neurons was higher than the calretinin+ subtype, which was a similar trend to in vivo. In addition, dopamine neuron subtype generation in vitro was not governed by exogenous sonic hedgehog and fibroblast growth factor 8. In the second study, we developed a scaffold-free, 3D neural spheroid culture using primary rat cerebral cortex cells. Cortical cells in agarose hydrogels containing round-bottom microwells self-assembled into spheroids. The 3D neural spheroids recapitulated key in vivo-like features including 3D neuronal and astroglial network formation, extracellular matrix production, electrical activity, circuitry formation via excitatory and inhibitory synapses, and brain-like mechanical properties. These physiologically relevant characteristics make these spheroids an effective in vitro model for the CNS. In the third study, we expanded the 3D culture technique to develop an in vitro model for stem cell transplantation to the CNS. To represent the host CNS tissue in vitro, trampoline-shaped 3D microtissues were fabricated using cortical cells. Neural stem cells (NSCs) in the form of spheroids were placed in the center of the CNS host trampoline microtissues, and NSC behavior in this transplantation model construct was monitored. In a proof-of-concept experiment, we utilized this model to test the effect of fibroblast growth factor 2 on NSC migration. Based on the microtissue characterization results, this 3D in vitro transplantation model has the potential to generate more translatable results than 2D co-culture platforms. These works together contribute to a deeper understanding of in vitro generation of neurons and provide a novel in vitro 3D engineered CNS model for the investigation of cell-based therapies.
Thesis (Ph.D. -- Brown University (2015)


Dingle, Yu-Ting Liu, "In Vitro Modeling of the Central Nervous System: Towards Optimized Cell-based Therapies" (2015). Biomedical Engineering Theses and Dissertations. Brown Digital Repository. Brown University Library.