Assessment of magnetic particles for neural stem cell-based therapies

Transplantation of genetically engineered neural stem cells (NSCs) into sites of central nervous system (CNS) disease/injury is a promising strategy to promote repair of damaged tissue. However, translating this strategy into the clinic requires several challenges to be overcome including facilitating ‘combinatorial therapy’ (achieving multiple therapeutic goals – essential in CNS injury/disease). Nanotechnologies are emerging as multifunctional platforms capable of meeting this requirement. For example, magnetic particles (MPs) and implantable hydrogels offer several biomedical advantages for transplant populations, including: safe genetic manipulation; non-invasive cell tracking, via MRI; and safe and efficient accumulation of cells at sites of injury. However, the use of these nanotechnologies remains to be explored in detail for NSC transplantation therapies.\ud \ud In this thesis, it is shown that MPs can mediate gene delivery to NSCs grown as neurospheres and monolayers with the most efficient transfection efficiencies achieved using oscillating magnetofection protocols (9.4% and 32.2% respectively). In both culture systems, developed protocols had no effect on key regenerative properties of NSCs such as cell viability, proliferation,\ud stemness and differentiation. Further, ‘magnetofected’ monolayer NSCs were shown to have survived and differentiated in a cerebellum slice model acting as host tissue, indicating safety of the procedures. It was also shown that assessing procedural safety and extent of transfection of magnetofection protocols may be feasible by employing mass spectrometry and proteomics analysis.\ud \ud It was also found that tailored enhancement of particle magnetite content offers a means to efficiently label NSCs, up to a maximum of 95.8%. Labelling procedures had no effect on cell viability, proliferation, stemness or differentiation. In addition, labelled cells could survive and differentiate in a slice model of spinal cord injury indicating safety of the labelling procedures. Functional labelling was also demonstrated by magnetic capture of labelled cells in an in vitro flow system.\ud \ud Hydrogels offer major advantages for delivery of transplant populations into injury sites. Here it was shown that an intraconstruct genetic engineering approach was feasible for NSCs cultured with a clinically translatable, collagen hydrogel system. Magnetofection protocols safely increased MP mediated transfection of NSCs grown in ‘2-D’ and ‘3-D’ hydrogel cultures