Nizamudeen, Zubair Ahmed
(2019)
Investigating the therapeutic potential of adult mouse cerebellum derived neural stem cells and the morphological characterisation of extracellular vesicles.
PhD thesis, University of Nottingham.
Abstract
Neural stem cells (NSCs) in the adult mammalian brain have been identified in the sub-ventricular zone (SVZ) of the lateral ventricles (LV) and the sub-granular zone (SGZ) of the hippocampus. Recently, the Bergmann glia (BG) of the adult mammalian cerebellum have been proposed as a NSC population of the adult brain. However, their regenerative potential is largely unclear. This project aims to investigate the properties of LV-derived and cerebellum-derived NSCs from the adult mouse brain by developing and optimising protocols to (a) differentiate primary NSCs, and (b) characterise extracellular vesicles (EV) secreted by NSCs.
The first results chapter focuses on testing a clinical drug called fasudil for neural differentiation in vitro. Fasudil is a a ROCK (Rho associated kinase) inhibitor which has been recently shown to differentiate C17.2 cell line. Here, the results show that fasudil can promote radial-like gliogenesis in primary LV-NSCs with increase in radial-like movement, radial glial markers GFAP and Sox2, and reduction in the neuronal marker βIII-tubulin. Moreover, this effect was consistent in multiple culture conditions including changes in serum concentration, substrate coating, and the batch of primary NSCs. Fasudil-based medium was thus used to study in vitro differentiation of primary NSCs.
The second results chapter focuses on developing an innovative tool to characterise EVs secreted by stem cells by using direct stochastic optical reconstruction microscopy (d-STORM- a super resolution imaging technique), and a lipid-based dye called DiD. The result show that the d-STORM approach was a reliable and a rapid alternative to conventional techniques used for EV characterisation including electron microscopy (EM), nanoparticle tracking analysis (NTA), and tunable resistive pulse sensing (TRPS). d-STORM provided superior sensitivity (20 nm to 30 nm) and speed (<1 hour) for analysing the EV size and count. The approach was further demonstrated to be useful to study intracellular uptake and trafficking of EVs in neural cells.
The third results chapter focuses on using the tools developed in the previous results chapters, including the fasudil-based NSC differentiation protocol and the d-STORM-based EV characterising approach, to investigate the NSC properties of BG isolated from the adult mouse cerebellum. Results showed that BG-NSCs can multiply and differentiate into neurons (expressing βIII-tubulin) and glia (expressing GFAP, S100b), similar to LV-NSCs. Image analysis of immunostained cerebellum sections from pcd (Purkinje cell degeneration) mutant mice model showed that BG increase in number during late stages of neurodegeneration. It was found that BG-NSCs secreted EVs that promote neuronal growth in a 2-D neuronal culture system, similar to LV-NSCs. BG-NSCs also showed some distinctive features compared to LV-NSCs such as the difference in morphology at early passages during cell culture, longer doubling time, producing larger neuronal bodies, expressing Calbindin in neural extensions, and secretion of 10-times more EVs per cell.
The fourth result chapter focuses on discovering the undesired effects of low-power sonication during EV analysis for future refinement of EV processing protocols. Results show that low-power sonication induces changes in size distribution, surface membrane properties and its functional effect including cellular uptake. Importantly, by comparing sonicated vs non-sonicated EVs in a 2-D neuronal culture system, it is shown that sonication of EVs is necessary for the neuronal outgrowth promoted by NSC derived EVs observed in the previous chapter. This chapter hence provides insights into the future considerations during EV research.
Taken together, this project provides a deeper understanding into NSC properties of BG, which was investigated by developing protocols to study cellular differentiation and extracellular vesicle characterisation.
Actions (Archive Staff Only)
|
Edit View |