UCLA

The central nervous system (CNS) consists of neurons and glia. In the healthy CNS, neurons form connected networks and oligodendrocytes, a type of glial cell, insulate and support the neuronal functions. When a spinal cord injury (SCI) occurs, a second type of glial cell, astrocytes infiltrate the injury site and cause a cascade of changes1. As a result, SCI often leads to permanent motor and sensory loss below the injury site2. In addition, the individual is faced with a lifetime of medical care ranging from $2.3-4.7 billion3. Around 300,000 people are currently living with SCI in the United States, with around 17,7000 new cases per year3. This creates a need and opportunity for restorative, rather than assistive, care.

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To repair the spinal cord, there are several considerations, including but not limited to, prevention or reversal of the glial scar formation, introduction of exogenous neural cells and stimulation of the repair process of severed axons. To produce lineages of cells that can be used for introduction at the SCI site, it is necessary to culture them first in vitro. Neural stem/progenitor cells (NS/PCs) are pluripotent precursor cells that can differentiate into neurons and glia and be cultured outside of the body4. Cells have been cultured in 2D environments for over a century5; however, technological advances of the late 19th century have provided new techniques to culture cells in a more biomimetic, 3D microenvironment6,7. These 3D microenvironments provide a medium to reliably culture cells into desired lineages, such as populations of neurons and oligodendrocytes. This research focuses on creating hyaluronic acid (HA)-based hydrogels that serve as 3D microenvironments for cultured NS/PCs in which they can proliferate and differentiate. HA is very biocompatible since it is naturally abundant within the human body, including in the spinal cord, as a key component of the extracellular matrix (ECM)8. One benefit of HA-based hydrogels is their tunability for different applications9. As shown in this research, the chemical and mechanical properties can be manipulated while maintaining the structural integrity of the hydrogel. Specifically, the HA concentration of the hydrogels can be independently assessed to better understand the interactions of HA with the encapsulated cells at different concentrations. NS/PC mechanostranduction of their microenvironment has been shown to produce signaling pathways within the cells with potential consequences on cell survival and growth10 so, another tunable parameter explored in this research was how NS/PCs respond to the stiffness of the hydrogels. HA has also been shown to reduce glial scar thickness by decreasing the number of astrocytes present after SCI11. As indicated, these HA-based hydrogels demonstrate several

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advantages as a vehicle for application of differentiated neural cells to the SCI site. HA provides a hydrated matrix in which cells can migrate, differentiate and communicate with cell membrane receptors on neural cells, such as CD44, RHAMM and ICAM-1 to elicit cascade effects such as morphogenesis, inflammation, wound repair and metastasis9. When HA receptors are bound they also cause a relay of biochemical and mechanical information about the microenvironment to effect cascades of intracellular changes12. Thus, it is important to understand and dissociate the effects of mechanical (stiffness) and chemical (HA concentration) properties of the hydrogel microenvironment to elucidate the distinct and/or synergistic effects. The hydrogels produced from this research aimed to test the effects of three different HA concentrations (0.1 wt%, 0.5 wt% and 1 wt%) on the survival, proliferation and differentiation of encapsulated NS/PCs. Another aspect of this project was to test varied mechanical properties of the hydrogels to determine the effect on encapsulated cells. Soft (200 Pascal), medium (400 Pascal) and stiff (800 Pascal) environments were compared, with values closely matched to the elasticity of biological nerve ECM which ranges from 0.5-1 kPa13. A 2D control was also compared to the hydrogel conditions to determine relative cell survival, proliferation and differentiation. Ultimately, this research is useful because it provides the opportunity to understand how the 3D environment alone (relatively no HA), the concentration of HA, and a softer vs. stiffer environment affects NS/PC proliferation and differentiation. The long-term goal of this research is to produce a robust and biocompatible in vitro microenvironment that directs differentiation of an enriched population of specific neural cell types, neurons and oligodendrocytes. Future research can then assess the efficacy of injecting the encapsulated neural cells into the SCI site as an approach to therapeutic repair.