Imaging Autophagy in hiPSC-Derived Midbrain Dopaminergic Neuronal Cultures for Parkinson’s Disease Research

To appreciate the positive or negative impact of autophagy during the initiation and progression of human diseases, the isolation or de novo generation of appropriate cell types is required to support focused in vitro assays. In human neurodegenerative diseases such as Parkinson’s disease (PD), specific subsets of acutely sensitive neurons become susceptible to stress-associated operational decline and eventual cell death, emphasizing the need for functional studies in those vulnerable groups of neurons. In PD, a class of dopaminergic neurons in the ventral midbrain (mDANs) is affected. To study these, human-induced pluripotent stem cells (hiPSCs) have emerged as a valuable tool, as they enable the establishment and study of mDAN biology in vitro. In this chapter, we describe a stepwise protocol for the generation of mDANs from hiPSCs using a monolayer culture system. We then outline how imaging-based autophagy assessment methodologies can be applied to these neurons, thereby providing a detailed account of the application of imaging-based autophagy assays to human iPSC-derived mDAN

Surface functionalisation of nanodiamonds for human neural stem cell adhesion and proliferation.

Biological systems interact with nanostructured materials on a sub-cellular level. These interactions may govern cell behaviour and the precise control of a nanomaterial's structure and surface chemistry allow for a high degree of tunability to be achieved. Cells are surrounded by an extra-cellular matrix with nano-topographical properties. Diamond based materials, and specifically nanostructured diamond has attracted much attention due to its extreme electrical and mechanical properties, chemical inertness and biocompatibility. Here the interaction of nanodiamond monolayers with human Neural Stem Cells (hNSCs) has been investigated. The effect of altering surface functionalisation of nanodiamonds on hNSC adhesion and proliferation has shown that confluent cellular attachment occurs on oxygen terminated nanodiamonds (O-NDs), but not on hydrogen terminated nanodiamonds (H-NDs). Analysis of H and O-NDs by Atomic Force Microscopy, contact angle measurements and protein adsorption suggests that differences in topography, wettability, surface charge and protein adsorption of these surfaces may underlie the difference in cellular adhesion of hNSCs reported here

Transplantation of Specific Human Astrocytes Promotes Functional Recovery after Spinal Cord Injury

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system

Preparation and characterisation of poly(vinyl) alcohol (PVA)/starch (ST)/halloysite nanotube (HNT) nanocomposite films as renewable materials

Poly(vinyl) alcohol (PVA)/starch (ST) films (weight ratio: 80/20) were prepared using a solution casting method, in the presence of 30 wt% glycerol (GL) as a plasticiser. Halloysite nanotubes (HNTs) were used as relatively new clay nanofillers to PVA/ST/GL blends for more economical material packaging. HNTs at filler loadings of 0.25, 0.5, 1, 3 and 5 wt% were incorporated to enhance mechanical and thermal properties of resulting PVA/ST/HNT nanocomposites. The tensile strength of such nanocomposites was found to be improved by 20 and 3.4%, respectively, with the inclusion of 0.25 and 0.5 wt% HNTs as opposed to those of PVA/ST/GL blends. However, a decreasing strength trend was observed beyond the HNT loading of 0.5 wt% due to HNT agglomeration, as evidenced by relevant micrographs via scanning electron microscopy (SEM). However, Young’s modulus was enhanced by 148% with the addition of 1 wt% HNTs when compared with PVA/ST/GL blends. X-ray diffraction (XRD) analysis is indicative of slightly intercalated nanocomposite structures formed at low HNT loadings of 0.25–1 wt%. In general, the incorporation of HNTs improved the thermal stability of PVA/ST/GL blends by increasing melting and decomposition temperatures along with the reduction in weight loss