Analysis of In Vitro Differentiation Cues in Adult Mouse Neural Stem and Progenitor Cells
Ludlow, Katherine Alaine
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Neural stem and progenitor cells (NSPCs) from the adult subventricular zone hold therapeutic potential for regenerative medicine. Specifically, differentiated oligodendrocytes can aid in remyelinating denuded axons in demyelinative disorders or spinal cord injury. The excitatory neurotransmitter, glutamate, has disputed roles in this multi-step process. The goal of my project was to determine if glutamate has an effect on myelination, with the primary focus on the differentiation aspect of NSPCs to oligodendrocytes. Changes to the gene expression profile of NSPC culture in response to oligodendrocyte driving media, glutamate, differentiation and attachment was investigated using reverse transcriptase quantitative polymerase chain reaction (RTQPCR) with cell marker gene targets. The combination of growth factors (FGF2, PDGF, NT3) used to drive, or increase the propensity of the NSPC to differentiate towards oligodendrocytes, had no effect on the proportions of cell types present in differentiated cultures. Therefore, data from driven or non-driven cultures could be combined for the rest of the treatments. Glutamic acid (200μM -1 mM) did not affect the survival of NSPC cultures when growing as floating neurospheres or attached to the extracellular matrix substrate, Matrigel. In addition, glutamate (200μM) did not have a significant effect on the gene expression profile of differentiated cultures. The differentiation media, containing fetal bovine serum (FBS) and no growth factors, significantly affected gene expression and promoted the development of neuronal lineage cells from preceding precursors, while decreasing the proportions of glial cells over time. It is suggested that the decrease in the majority of cell markers could also be the result of a large cell die off from proliferation and subsequent confluency issues during the differentiation period. Cultures in regular media, but also attached to Matrigel-coated dishes, saw significant increases in NSC and glial markers, with possible inhibition of the neuronal differentiation favoured by spontaneous attachment. The cause of spontaneous attachment was undetermined and promoted the differentiation of NSPCs towards neuronal and oligodendrocyte lineage cells. Necessary improvements to the current experimental design, such as an optimal driving, and ideally, cell sorting protocol, would allow for more conclusive evidence to be presented for glutamate’s role on NSPC differentiation. Attempts were made to investigate glutamate’s role on myelination by observing migration of oligodendrocyte precursor cells (OPCs) using co-cultures of differentiated NSPCs with dorsal root ganglion neurons and visualization by immunocytochemistry. There were antibody specificity and technique issues, most of which have been optimized, but these experiments did not produce substantive results. An apparent lack of mature oligodendrocytes in these co-cultures prompted the need for differentiation experiments, which demonstrated the presence of mature oligodendrocytes over the differentiation period required for co-culture experiments. Conversely, the lack of effect observed for the driving media may have also contributed to the apparent insufficient oligodendrocytes present for immunocytochemistry analysis. Therefore, improvements are suggested for the differentiation protocols, which would increase the ability to specifically investigate glutamate’s effect on myelination and migration.