Analysis of the Differentiation of Adult Neural Stem and Progenitor Cells Post Treatment with the ASH1 Transcription Factor

Abstract

Adult neural stem and progenitor cells hold the potential to regenerate lost tissue following injury or disease to the central nervous system. Before these cells can be used to regenerate tissue, they must be instructed to differentiate into the appropriate cell type. The objectives of this thesis were to investigate the differentiation induced from the proneural transcription factor ASH1 with an intracellular delivery mechanism in adult neural stem and progenitor cells cultured in vitro and to develop an in-house biosensor capable of detecting the neurotransmitter glutamate from non-myelinated axons. It was demonstrated that the addition of the cell permeable ASH1 promoted neurogenesis in neural stem and progenitor cells after two weeks of cell culture. Patch-clamp electrophysiology and immunocytochemistry were used to verify that the cells were differentiating into neurons. Although the cells did not develop into mature neurons capable of generating action potentials, they showed neuronal characteristics including the expression of neurofilament and the presence of voltage-gated Na+ channels. In addition, it was shown, using patch-clamp electrophysiology on HEK293T cells transduced to express the glutamate ionotropic receptor GRIK3, that glutamate can be detected in the bath solution. This work shows that HASH1 can be used to induce neuronal differentiation in adult neural stem and progenitor cells in vitro and the inhouse biosensor can be used to detect potential glutamate release from axons of neurons generated by ASH1.