1. To examine the electrophysiology and synaptic biology of neurons differentiated using our lab’s human induced pluripotential stem cell (iPS) protocols in patients with and without Alzheimer’s disease.
2. To translate our successful protocol for generating neurons from adult canine genetically unmodified Skin?derived Neuroprecursors (SKiNPs) to an efficient, high?yield adult human protocol
3. To compare human SKiNPs with human iPS results
4. Overall, to evaluate the potential of both iPS? and SKiNP?based systems as cellular in vitro models for Alzheimer’s Disease
One of the main limitations for advancing our understanding of central nervous system (CNS) disorders is the necessary restriction on direct biopsy of brain tissue. Research on CNS disease is therefore mainly driven by developing animal models and examining post mortem human brain tissue, each of which has its own strengths and weaknesses.
The discovery of induced pluripotential stem (iPS) cells – which resemble embryonic stem cells yet are derived from genetic modification of adult somatic cells (usually fibroblasts from skin) – has opened the door to developing patient?specific cell lines of any tissue type, including neurons. In this fast moving field, researchers have already shown that iPS cell lines can be successfully grown from patients with genetically?determined neurodegenerative diseases, and that these cells recapitulate certain basic features of the disease in vitro.
As yet, there have been no documented studies of iPS?derived neurons in patients with familial or sporadic Alzheimer’s disease (AD). Beyond the simple cellular characteristics of these cells in comparison to control non?AD individuals, it is forecast that if these cells do capture important aspects of AD in situ pathophysiology, then more marked and salient differences may arise in terms of neuronal function. The main emphasis of this PhD will therefore be an examination of electrophysiological and synaptic biological differences between AD patient iPS cells and control cell lines. This part of the project will be well supported by our Stem Cell Laboratory’s published and successful human iPS protocols.
Whilst iPS cells have revolutionized the field, they also have some important limitations. iPS cells depend on the knock?in of one or more pluripotential genes, each of which may interact with the AD molecular regulatory network, and hence distort the validity of any resulting cellular model. iPS are a low efficiency system, requiring 1000s of iPS cells to yield one viable cell line, and thereafter their neuronal differentiation is low. Given their genetic modifications, iPS cells are also not safe to use in any future therapeutic application.
Our group has therefore optimized an efficient and high yield system of generating neurons from native (genetically unmodified) skin, through the isolation of skin?stem cells and redifferentiation into neuroprecursors. These cells, termed Skin derived NeuroPrecursors (SKiNPs), have been well characterised using donor adult canine tissue. The second major aim of this PhD will therefore be to translate our canine SKiNP technology to a successful human SKiNP protocol, and thereafter investigate AD SKiNP?derived neuronal electrophysiology and synaptic biology. These results will be compared to both control non?AD SKiNP as well as iPS findings. In this way, we will be able to evaluate whether a SKiNP?based cell model is both efficient and practical as well as providing a cell model of AD.
Altogether, this PhD will therefore result in a new understanding of AD by the examination and exploration of different cellular skin?based models. Future significance may include discovery of new AD pathophysiologic pathways, and application for drug screening, clinical diagnosis and prognostics.