Generation and investigation of neural microenvironment in health and disease

Abstract

Cells in our body are under the constant influence of intrinsic and extrinsic factors that modulate their behavior. Early on, starting during the development of the organism, growth factors, nutrients, oxygen, the extracellular matrix and contact with other cells affect essential processes such as proliferation, migration, differentiation and apoptosis. Later on, the same types of stimuli regulate tissue homeostasis and play important roles in disease onset. Although our understanding of the cellular microenvironment and its impact on the cells is deeper than ever before, we still do not fully understand the whole complexity behind it, making the need to not only study the microenvironment but also replicate it in vitro more pressing. Areas such as tissue engineering, disease modeling, drug testing and in vitro microphysiological systems are just some examples where new knowledge and tools in this field are necessary for progress.

This thesis reflects the path I took to first study and then to recreate some of the aspects of the neural microenvironment. Paper I is a study of the redox sensing co-repressor CtBP2. This protein is well known to integrate oxygen levels and regulate gene expression during development and disease. When knocked down in the mouse embryonic brain, normal cortex formation was disrupted, revealing an important role of CtBP2 in neural stem cell maintenance, differentiation, and migration. In Paper II, we focused on CtBP2 at the molecular level, studying it in vitro in rat neural stem cells (NSCs). By applying different metabolic conditions in vitro, we showed that hypoxia and 2-Deoxy-D-glucose increased acetylation of CtBP2 in proliferating NSCs. Additionally, 1% oxygen treatment resulted in altered homodimerization of CtBP2 showing that some aspects of the NSC environment are conveyed to post-translational modifications of CtBP2, an important step in its functional regulation.

Paper III explores the use of 3D bioprinting technology and a special material deposition technique – freeform reversible embedding of suspended hydrogels (FRESH) – to generate a relevant microenvironment for human neuroblastoma 3D cell culture. Using a Parameter Optimization Index (POI), we optimized sodium alginate (SA) printing parameters including extrusion pressure and speed. With this approach, we successfully printed human neuroblastoma cells in 3D encapsulated in SA, maintaining good cell viability and high print quality. Lastly, to find a more robust biomaterial for the in vitro microenvironment engineering for human neuroepithelial-like stem cells (NESCs), in Paper IV we investigated the cytocompatibility of these cells with vitronectin-modified recombinant spider silk (VN- NT2RepCT). Our study showed the necessity of spider silk functionalization with vitronectin to provide attachment for NESCs. This material successfully supported neural stem cell growth and proliferation, and further analysis of the morphology of focal adhesions revealed differences in cell attachment compared to control substrates. Our results suggest potential applications for the VN-NT2RepCT in tissue engineering, possibly including 3D bioprinting.