Florey Department of Neuroscience and Mental Health - Theses

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    Understanding neural development and the establishment of region-specific protocols for the differentiation of naive pluripotent stem cells
    Alsanie, Walaa Fahad ( 2016)
    Pluripotent stem cells (PSCs) have the potential to differentiate into the three germ lineages including ectoderm, and more specifically neuroectoderm, in an effort to generate different neuronal populations. These resultant neural progenitors and neurons can be utilized for various purposes, such as modeling development and/or disease in vitro, neural transplantation, as well as drug development and testing. Studies to date involving the neural differentiation of mouse PSCs have been hampered by the lack of highly defined protocols to generate specific neural populations. Hence, improved protocols to derive specific neural populations from PSCs are highly required. Chapters 3 and 4 of this PhD thesis focus on developing novel differentiation procedures to generate region-specific neurons (dorsal forebrain, ventral forebrain, ventral midbrain, hindbrain and medial ganglionic eminence) from naïve ground state PSCs. To begin, we demonstrate that the use of naïve mouse embryonic stem cells (mESCs), reflective of blastocyst pre-implantation pluripotent stem cells, improves neural differentiation in comparison to primed, postimplantation- like, mESCs. To direct the fate of neural progenitors, we have utilized a number of morphogens (such as SHH for ventralization and Wnts for caudalization) that are involved in mammalian neural development. Furthermore, the spatial and temporal expressions of the progenitors and mature neurons, derived using our novel differentiation systems, have confirmed their region-specific identity. Whilst these improved protocols enable the generation of regionally specified neural progenitors and neurons, more restricted balances between extrinsic morphogen gradients (such as those manipulated in chapters 3 and 4) together with specific intrinsic gene profiling results in the more specialized populations of neurons within each of these regions, such as the dopaminergic (DA) neurons within the ventral midbrain (VM). Despite decades of research into understanding the development of VM DA neurons, numerous key events involved in their specification and connectivity remain unknown. A recent microarray study within laboratory generated a gene list of novel genes involved in ventral midbrain. Included in this list, and examined in chapter 5 of this thesis is the role of the cell adhesion protein, Close homology to L1 (CHL1), in the birth and connectivity of midbrain dopamine neurons. Using VM primary cultures and Chl1 deficient mice, the results from this chapter show the diverse roles of both soluble and membrane-bound CHL1 in migration of DA progenitors, their differentiation and finally their connectivity. We identify that in these contexts, CHL1 acts through homophilic (CHL1-CHL1) interactions between the DA neuron and the local environment. These findings provide the first reports of a cell adhesion protein in the development of VM DA neurons. The results from these experiments could (i) improve the derivation of specific neuronal populations, hence, improving their use in drug testing, transplantation and disease modeling. For example, MGE-specific progenitors and mature neurons, derived using our protocol, could be used to study epilepsy or could be used for drug screening and development. In addition, transgenic lines could be differentiated using our protocols to region-specific neurons to understand their development in vitro. The results from these experiments could also (ii) expand our knowledge regarding the development of the VM DA neurons. By understanding the roles of CHL1 in VM DA development, we could use this knowledge to enhance the generation of VM DA neurons from human PSCs. Additionally, CHL1 could be utilized to enhance the outcomes of cellular replacement therapies for different neurological disorders, such as Parkinson’s disease.