Chitosan-Based Biomaterials for Renewal and Differentiation of Human Neural Stem Cells

Abstract

Human neural stem cells (hNSCs) are critical for regenerative medicine and disease modeling due to their self-renewal and ability to differentiate into neurons, astrocytes, and oligodendrocytes. These traits make hNSCs ideal for repairing neural tissue and studying neurodegenerative disorders like Alzheimer's and Parkinson's, which are increasingly prevalent in aging populations and carry significant socioeconomic costs. Stem cell-based strategies offer transformative potential for therapeutic development and mechanistic studies. However, hNSC applications are hindered by current in vitro culture systems. Conventional 2D platforms, often using murine-derived extracellular matrix (ECM) coatings like Geltrex or Matrigel, fail to mimic the brain's 3D microenvironment. These materials are not chemically defined or xeno-free, posing regulatory and safety challenges for clinical translation. Cells grown on such substrates require extensive testing to meet current Good Manufacturing Practice (cGMP) standards, increasing costs and complexity. Moreover, 2D systems lack physiological relevance, contributing to the high failure rate of drug candidates in clinical trials. This dissertation addresses these challenges through a systematic exploration of chitosan-based biomaterials as a versatile, next-generation platform for hNSC expansion, maintenance, differentiation, and disease modeling. Chitosan, a biocompatible and biodegradable polysaccharide derived from chitin, closely mimics the glycosaminoglycans in the brain's ECM, offering a biomimetic substrate for neural cell culture. Its ability to be processed under mild conditions into various morphologies, such as films, scaffolds, or hydrogels, and chemically modified to tune mechanical and biological properties makes it an ideal candidate for advanced stem cell engineering applications.The research began with the fabrication and characterization of composite thin films made from chitosan, alginate, and hyaluronic acid. These films were designed to optimize key parameters, including surface hydrophilicity, mechanical stiffness, nanoscale topography, and electrostatic interactions with hNSCs. By systematically varying polymer ratios, we found that pure chitosan films provided the most supportive environment for maintaining hNSC multipotency over a 4-day culture period, as evidenced by sustained expression of stemness markers (SOX2, nestin, Ki67, and SOX1) assessed via an alamarBlue assay, immunocytochemistry, and flow cytometry. These findings established pure chitosan as the foundational material for subsequent 3D scaffold development. To address the limitations of 2D cultures and enable scalable production of functional stem cells, we designed 3D porous chitosan scaffolds optimized for dynamic culture conditions. Using an orbital shaker model to simulate perfusion and mechanical agitation, we demonstrated that these scaffolds maintained structural integrity under fluid shear stress while supporting hNSC viability and function. The porous architecture facilitated enhanced nutrient and oxygen diffusion, promoting physiologically relevant cell–cell and cell–matrix interactions. Compared to static 2D controls, these scaffolds significantly improved hNSC proliferation and multipotency, as measured by alamarBlue assay, live/dead staining, and SOX2, nestin, SOX1, and PAX6 staining. This system supported long-term culture of hNSCs (over 21 days). Scaffolds with higher chitosan content (e.g., 4% w/v) exhibited superior mechanical resilience, making them well-suited for bioprocessing applications in large-scale cell manufacturing. Recognizing the need for controlled, scalable stem cell production, we developed a custom, modular bioreactor system capable of supporting both macro- and microscale cultures. The bioreactor featured real-time, in-line monitoring of critical parameters, pH, dissolved oxygen, and CO₂, using integrated sensors connected to a microcontroller-based interface for precise environmental control. This system supported the culture of hNSCs for 7 days, as well as other cell types such as suspension-grown T cells and adherent tumor cell lines, demonstrating its broad applicability. The use of cost-effective, off-the-shelf components enhances the system's accessibility for academic research and its potential for adoption in translational settings, such as cGMP-compliant cell production facilities. We then investigated chitosan scaffolds as a platform for directing hNSC differentiation into cortical neurons. Various surface modifications, including Geltrex coatings, medium conditioning with neurotropic factors (e.g., BDNF, GDNF), and pre-seeding techniques, were evaluated for their impact on lineage commitment. Pre-seeded scaffolds outperformed 2D controls, achieving higher neuronal yields and faster differentiation (within 14 days), with reduced astrocytic phenotypes, as confirmed by flow cytometry, immunocytochemistry, RT-qPCR analyses. These results highlight the scaffolds' ability to create a 3D microenvironment that closely mimics in vivo neural development. To validate the platform's translational potential, we cultured human induced pluripotent stem cell (hiPSC)-derived neural progenitors from healthy donors and Alzheimer's disease (AD) patients on aligned porous chitosan scaffolds. These scaffolds supported the formation of functional 3D neural networks, with AD-derived cultures exhibiting disease-specific hallmarks, including amyloid-beta peptide accumulation and dysregulated tau-related gene expression (e.g., MAPT, PSEN1). These phenotypes were quantified using ELISA and RNA sequencing, confirming the platform's utility as a scalable, physiologically relevant model for neurodegenerative disease research and preclinical drug screening. In conclusion, chitosan-based biomaterials offer a chemically defined, xeno-free, and scalable solution to key challenges in hNSC culture, differentiation, and disease modeling. By meeting cGMP regulatory standards and enabling the transition from static cultures to dynamic bioreactor systems, these platforms support both research and therapeutic applications. This work advances the field of regenerative medicine and provides a robust, biomimetic tool for studying neurodegenerative disease mechanisms, paving the way to produce high-quality, clinically relevant neural cells.