• Alignment of multiple glial cell populations in 3D nanofiber scaffolds: toward the development of multicellular implantable scaffolds for repair of neural injury

      Weightman, Alan P.; Jenkins, Stuart I.; Pickard, Mark R.; Chari, Divya M.; Yang, Ying; Keele University, United Kingdom (Elsevier, 2014-02)
      Non-neuronal cells of the central nervous system (CNS), termed "neuroglia," play critical roles in neural regeneration; therefore, replacement of glial populations via implantable nanofabricated devices (providing a growth-permissive niche) is a promising strategy to enhance repair. Most constructs developed to date have lacked three-dimensionality, multiple glial populations and control over spatial orientations, limiting their ability to mimic in vivo neurocytoarchitecture. We describe a facile technique to incorporate multiple glial cell populations [astrocytes, oligodendrocyte precursor cells (OPCs) and oligodendrocytes] within a three-dimensional (3D) nanofabricated construct. Highly aligned nanofibers could induce elongation of astrocytes, while OPC survival, elongation and maturation required pre-aligned astrocytes. The potential to scale-up the numbers of constituent nanofiber layers is demonstrated with astrocytes. Such complex implantable constructs with multiple glial sub-populations in defined 3D orientations could represent an effective approach to reconstruct glial circuitry in neural injury sites.
    • Endocytotic potential governs magnetic particle loading in dividing neural cells: studying modes of particle inheritance

      Tickle, Jacqueline A.; Jenkins, Stuart I.; Polyak, Boris; Pickard, Mark R.; Chari, Divya M.; Keele University, United Kingdom; Drexel University College of Medicine, Philadelphia, USA (Future Medicine, 2016-01-10)
      AIM: To achieve high and sustained magnetic particle loading in a proliferative and endocytotically active neural transplant population (astrocytes) through tailored magnetite content in polymeric iron oxide particles. MATERIALS & METHODS: MPs of varying magnetite content were applied to primary-derived rat cortical astrocytes ± static/oscillating magnetic fields to assess labeling efficiency and safety. RESULTS: Higher magnetite content particles display high but safe accumulation in astrocytes, with longer-term label retention versus lower/no magnetite content particles. Magnetic fields enhanced loading extent. Dynamic live cell imaging of dividing labeled astrocytes demonstrated that particle distribution into daughter cells is predominantly 'asymmetric'. CONCLUSION: These findings could inform protocols to achieve efficient MP loading into neural transplant cells, with significant implications for post-transplantation tracking/localization.
    • Identifying the cellular targets of drug action in the central nervous system following corticosteroid therapy

      Jenkins, Stuart I.; Pickard, Mark R.; Khong, Melinda; Smith, Heather L.; Mann, Carl L. A.; Emes, Richard D.; Chari, Divya M.; Keele University, University of Nottingham, University Hospital of North Staffordshire NHS Trust, United Kingdom (American Chemical Society, 2014-01-15)
      Corticosteroid (CS) therapy is used widely in the treatment of a range of pathologies, but can delay production of myelin, the insulating sheath around central nervous system nerve fibers. The cellular targets of CS action are not fully understood, that is, "direct" action on cells involved in myelin genesis [oligodendrocytes and their progenitors the oligodendrocyte precursor cells (OPCs)] versus "indirect" action on other neural cells. We evaluated the effects of the widely used CS dexamethasone (DEX) on purified OPCs and oligodendrocytes, employing complementary histological and transcriptional analyses. Histological assessments showed no DEX effects on OPC proliferation or oligodendrocyte genesis/maturation (key processes underpinning myelin genesis). Immunostaining and RT-PCR analyses show that both cell types express glucocorticoid receptor (GR; the target for DEX action), ruling out receptor expression as a causal factor in the lack of DEX-responsiveness. GRs function as ligand-activated transcription factors, so we simultaneously analyzed DEX-induced transcriptional responses using microarray analyses; these substantiated the histological findings, with limited gene expression changes in DEX-treated OPCs and oligodendrocytes. With identical treatment, microglial cells showed profound and global changes post-DEX addition; an unexpected finding was the identification of the transcription factor Olig1, a master regulator of myelination, as a DEX responsive gene in microglia. Our data indicate that CS-induced myelination delays are unlikely to be due to direct drug action on OPCs or oligodendrocytes, and may occur secondary to alterations in other neural cells, such as the immune component. To the best of our knowledge, this is the first comparative molecular and cellular analysis of CS effects in glial cells, to investigate the targets of this major class of anti-inflammatory drugs as a basis for myelination deficits.
    • An in vitro spinal cord injury model to screen neuroregenerative materials

      Weightman, Alan P.; Pickard, Mark R.; Yang, Ying; Chari, Divya M.; Keele University (Elsevier, 2014-01-29)
      Implantable 'structural bridges' based on nanofabricated polymer scaffolds have great promise to aid spinal cord regeneration. Their development (optimal formulations, surface functionalizations, safety, topographical influences and degradation profiles) is heavily reliant on live animal injury models. These have several disadvantages including invasive surgical procedures, ethical issues, high animal usage, technical complexity and expense. In vitro 3-D organotypic slice arrays could offer a solution to overcome these challenges, but their utility for nanomaterials testing is undetermined. We have developed an in vitro model of spinal cord injury that replicates stereotypical cellular responses to neurological injury in vivo, viz. reactive gliosis, microglial infiltration and limited nerve fibre outgrowth. We describe a facile method to safely incorporate aligned, poly-lactic acid nanofibre meshes (±poly-lysine + laminin coating) within injury sites using a lightweight construct. Patterns of nanotopography induced outgrowth/alignment of astrocytes and neurons in the in vitro model were strikingly similar to that induced by comparable materials in related studies in vivo. This highlights the value of our model in providing biologically-relevant readouts of the regeneration-promoting capacity of synthetic bridges within the complex environment of spinal cord lesions. Our approach can serve as a prototype to develop versatile bio-screening systems to identify materials/combinatorial strategies for regenerative medicine, whilst reducing live animal experimentation.
    • Influence of Amplitude of Oscillating Magnetic Fields on Magnetic Nanoparticle-Mediated Gene Transfer to Astrocytes

      Tickle, Jacqueline A.; Jenkins, Stuart I.; Pickard, Mark R.; Chari, Divya M.; Keele University, United Kingdom (World Scientific, 2014-08-07)
      Functionalized magnetic nanoparticles (MNPs) are emerging as a major nanoplatform for regenerative neurology, particularly as transfection agents for gene delivery. Magnetic assistive technology, particularly the recent innovation of applied oscillating magnetic fields, can significantly enhance MNP-mediated gene transfer to neural cells. While transfection efficiency varies with oscillation frequency in various neural cell types, the influence of oscillation amplitude has not yet been investigated. We have addressed this issue using cortical astrocytes that were transfected using MNPs functionalized with plasmid encoding a reporter protein. Cells were exposed to a range of oscillation amplitudes (100–1000 μm), using a fixed oscillation frequency of 1 Hz. No significant differences were found in the proportions of transfected cells at the amplitudes tested, but GFP-related optical density measurements (indicative of reporter protein expression) were significantly enhanced at 200 μm. Safety data show no amplitude-dependent toxicity. Our data suggest that the amplitude of oscillating magnetic fields influences MNP-mediated transfection, and a tailored combination of amplitude and frequency may further enhance transgene expression. Systematic testing of these parameters in different neural subtypes will enable the development of a database of neuro-magnetofection protocols — an area of nanotechnology research where little information currently exists.
    • The influence of nicotinamide on the development of neurons

      Griffin, Sile; Pickard, Mark R.; Hawkins, Clive P.; Williams, Adrian C.; Chari, Divya M.; Fricker, Rosemary; Orme, Rowan P.; Keele University, University Hospital of North Staffordshire NHS Trust, University of Birmingham, United Kingdom (2014-09-09)
      A major challenge in translating the promise of stem cell therapies to treat a myriad of neurodegenerative disorders is to rapidly and efficiently direct pluripotent stem cells to generate differentiated neurons. The application of active vitamin metabolites known to function in embryonic development and maintenance in the adult brain such as retinoic acid (vitamin A), ascorbic acid (vitamin C) and calcitriol (vitamin D3) have proven effective in current in-vitro differentiation protocols. Therefore, in this study we investigated whether the biologically active vitamin B3 metabolite, nicotinamide could enhance the differentiation of mouse embryonic stem cells, cultured as monolayers, into mature neurons at either early or late stages of development. Interestingly, nicotinamide elicited a dose-responsive increase in the percentage of neurons when added at an early developmental stage to the cells undergoing differentiation (days 0–7). Nicotinamide (10 mM) increased the proportion of β-III tubulin positive neurons by two fold and concomitantly decreased the total number of cells in culture, measured by quantification of 4′, 6-diamidino-2-phenylindole positive cells. This effect could result from induction of cell-cycle exit and/or selective cell death in non-neural populations. Higher levels of nicotinamide (20 mM) induced cytoxicity and cell death. This study supports previous evidence that vitamins and their metabolites can efficiently direct stem cells into neurons. Current work is focusing on the effect of nicotinamide on the process of neural induction and whether nicotinamide influences the generation of particular neuronal subtypes implicated in neurodegenerative diseases, specifically focusing on midbrain dopamine neurons; towards a therapy for Parkinson's disease.
    • Magnetic nanoparticle-mediated gene delivery to two- and three-dimensional neural stem cell cultures: magnet-assisted transfection and multifection approaches to enhance outcomes

      Pickard, Mark R.; Adams, Christopher F.; Chari, Divya M.; University of Chester; Keele University (Wiley, 2017-02-02)
      Neural stem cells (NSCs) have high translational potential in transplantation therapies for neural repair. Enhancement of their therapeutic capacity by genetic engineering is an important goal for regenerative neurology. Magnetic nanoparticles (MNPs) are major non-viral vectors for safe bioengineering of NSCs, offering critical translational benefits over viral vectors, including safety, scalability, and ease of use. This unit describes protocols for the production of suspension (neurosphere) and adherent (monolayer) murine NSC cultures. Genetic engineering of NSCs with MNPs and the application of 'magnetofection' (magnetic fields) or 'multifection' (repeat transfection) approaches to enhance gene delivery are described. Magnetofection of monolayer cultures achieves optimal transfection, but neurospheres offer key advantages for neural graft survival post-transplantation. A protocol is presented which allows the advantageous features of each approach to be combined into a single procedure for transplantation. The adaptation of these protocols for other MNP preparations is considered, with emphasis on the evaluation of procedural safety.
    • Using magnetic nanoparticles for gene transfer to neural stem cells: stem cell propagation method influences outcomes

      Pickard, Mark R.; Adams, Christopher F.; Barraud, Perrine; Chari, Divya M.; Keele University, United Kingdom; University of Cambridge, United Kingdom (Multidisciplinary Digital Publishing Institute, 2015-04-24)
      Genetically engineered neural stem cell (NSC) transplants offer a key strategy to augment neural repair by releasing therapeutic biomolecules into injury sites. Genetic modification of NSCs is heavily reliant on viral vectors but cytotoxic effects have prompted development of non-viral alternatives, such as magnetic nanoparticle (MNPs). NSCs are propagated in laboratories as either 3-D suspension "neurospheres" or 2-D adherent "monolayers". MNPs deployed with oscillating magnetic fields ("magnetofection technology") mediate effective gene transfer to neurospheres but the efficacy of this approach for monolayers is unknown. It is important to address this issue as oscillating magnetic fields dramatically enhance MNP-based transfection in transplant cells (e.g., astrocytes and oligodendrocyte precursors) propagated as monolayers. We report for the first time that oscillating magnetic fields enhanced MNP-based transfection with reporter and functional (basic fibroblast growth factor; FGF2) genes in monolayer cultures yielding high transfection versus neurospheres. Transfected NSCs showed high viability and could re-form neurospheres, which is important as neurospheres yield higher post-transplantation viability versus monolayer cells. Our results demonstrate that the combination of oscillating magnetic fields and a monolayer format yields the highest efficacy for MNP-mediated gene transfer to NSCs, offering a viable non-viral alternative for genetic modification of this important neural cell transplant population.