Utilizing 3D Hydrogel Systems to Evaluate the Proliferation and Differentiation of Oligodendrocyte Progenitor Cells in the Presence of Electrospun Fibers

Demyelinated neural environments, such as those affected by multiple sclerosis (MS) or traumatic brain injury, require an increased presence of, and remyelination by, oligodendrocyte cells (OLs) to repair the damage. These OLs stem from lineage-restricted oligodendrocyte progenitor cells (OPCs), which can both proliferate and differentiate. The Lampe Group commonly encapsulates OPCs in extracellular matrix-mimicking 3D hydrogel systems to observe cell response to stimuli, where lower stiffness gels are conducive to higher rates of viability and proliferation¹. We have observed that the addition of axon–mimicking electrospun fibers in norbornene-functionalized hyaluronic acid (NorHA) gels leads to increased OPC process extension, a preliminary indication of differentiation toward OLs. This contrasts the circular morphology of cells found in similar gels that do not contain fibers. Since both the proliferation and differentiation of OPCs are integral to the remyelination process, we aim to create environments to study and maximize each rate. In the current hydrogel system, we hypothesize that the energy devoted to morphology changes in the presence of fibers reduces the proliferation rate. Over a 7-day period, NorHA gels containing 1% methacrylated hyaluronic acid (MeHA) electrospun fibers and gels containing no fibers were compared using EdU and DAPI stains. When cells synthesize DNA during proliferation, the fluorescent EdU stain is incorporated into the new strands. DAPI is a fluorescent stain that binds to all DNA, fluorescently labeling each cell to give a total cell count. Ratios of the two stains were utilized to measure average proliferation per cell. OPCs encapsulated in gels without fibers consistently proliferated at higher rates than OPCs in the fiber-containing gels (roughly 2-fold higher, day 7), which instead extended more and longer branches. This indicates that the presence of axonal-like fibers increases OPC differentiation into OLs but also decreases proliferation. To confirm trends seen in the former analysis method and to ensure no technique bias, the ratio of EdU to DAPI signal area was used as an additional measure of proliferation rates. The results of this analysis also allowed the conclusion that proliferation decreased while process extension increased with the addition of fibers.

References:
1. Unal, D. B., Caliari, S. R., & Lampe, K. J. (2020). 3D Hyaluronic Acid Hydrogels for Modeling Oligodendrocyte Progenitor Cell Behavior as a Function of Matrix Stiffness. doi:10.1101/2020.04.01.020412