Characterizing effects of puromycin selection on enrichment of astrocyte subtypes and extracellular matrix (ECM) generation with ECM incorporation in hyaluronic-acid (HA)-based hydrogels for axon growth of V2a interneurons

Abstract

Astrocyte subtypes are key cellular players to study in vitro for identifying specific cues that increase axon growth of neuron populations in potential translation to develop a scalable astrocyte-derived solution for axon regeneration post SCI. I characterized the use of transgenic puromycin selectable cell lines to enrich protoplasmic and fibrous astrocytes differentiated from mouse embryonic stem cells (mESCs) through viability and glutamate uptake assays, immunocytochemistry, flow cytometry, quantitative polymerase chain reaction (qPCR), and calcium imaging. It was demonstrated that selected astrocyte subtypes maintain low levels of other cell types (mature neurons and oligodendrocytes and undifferentiated stem cells) and high levels of astrocyte markers with functionality by glutamate uptake, inflammatory response, and calcium transients. Furthermore, selected protoplasmic astrocyte components, specifically ECM, demonstrated significant increases in axon growth of mESC-derived V2a interneurons, a critical excitatory neuron population found in grey matter of spinal cord, compared to selected fibrous astrocytes in both 2D and 3D HA-based hydrogel setting. With bulk-RNA sequencing of astrocyte subtypes and proteomic analysis of selected astrocyte subtype ECM, selected protoplasmic astrocytes demonstrated upregulation of critical adhesion and ECM-related genes and proteins that could contribute to the positive impact on axon growth. Based on the criteria of protein availability and manufacturing, 5 selected protoplasmic ECM proteins at defined combinations and concentrations (based on design of experiments- DoE approach) in HA-based hydrogel were shown to significantly increased axon growth of V2a interneurons compared to selected protoplasmic ECM hydrogel. Overall, this thesis demonstrates the importance of studying in vitro properties of astrocyte subtypes for axon growth and the use of astrocyte-derived factors in developing a scalable, cell-free permissive biomaterial for supporting axon regeneration in future SCI applications.