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Induced pluripotent stem cells have been for the first time derived from human fibroblasts by ectopic expression of four transcription factors - OCT4, SOX2, KLF4, and c-MYC (OSKM). To date, pluripotent reprogramming is a very unreliable and inefficient process with an underlying mechanism that remains elusive. In terminally differentiated cells such as fibroblasts, pluripotency genes are transcriptionally silent and found within closed chromatin regions. The high nucleosome density of these silent chromatin regions occludes the access of most DNA-binding proteins to their gene targets. Previously, OSK have been shown to act as pioneer factors that can access closed chromatin by directly interacting with nucleosomes. To examine how OCT4 interacts with nucleosomes during reprogramming, I used cryogenic-electron microscopy (cryo-EM) and computational modelling to reveal the three-dimensional arrangement of OCT4 in complex with a nucleosome containing native Lin28B or Esrrb enhancer sequence, which are both targeted by OCT4 in early reprogramming. First, I have optimized native nucleosome reconstitution to make it suitable for cryo-EM studies. As a result, I present structural data of two native nucleosomes at low resolution as well as a high-resolution cryo-EM analysis of one native nucleosome. This has revealed regions within nucleosome with enhanced local DNA flexibility. Further computational modelling illustrates OCT4 binding at three different target sites on nucleosome. Together, these results propose that OCT4 adopts a specific conformation to recognise one part of its motif specifically using its POUS DNA-binding domain, while the other POUHD DNA-binding domain interacts with DNA non-specifically. This is consistent with the idea that OCT4 uses one of its two POU DNA-binding domains to bind a partial motif exposed on the nucleosome surface in a sequence-specific manner. Overall, revealing the structural features of OCT4-nucleosome complex could help us understand the molecular mechanism by which OCT4 engages its DNA binding sites in closed chromatin and elucidates changes in local chromatin landscape during cell-fate reprogramming.

Diabetic endotheliopathy is the main cause for impaired angiogenesis and reduced neovascularization that lead to microvascular injury and vascular complications. The pathogenesis for vascular complications arising from diabetes is complex. Elucidation of key underlying mechanisms will help the development of novel therapies and the discovery of potential biomarkers. The ability to generate functional endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) is a novel and powerful tool for cell-based therapies. Human iPSC-derived ECs (iPS-ECs) have a broad range of clinical applications including cell-based therapy, disease modelling and drug screening; they can be used in mechanistic studies towards the development of novel therapies and in the discovery of new biomarkers to be applied in regenerative medicine and treatment of diabetic vasculopathy. Here we utilize transcriptomic and proteomic technologies to assess patient-specific iPS-ECs from diabetic (DiPS-ECs) and non-diabetic (NiPS-ECS) donors in order to investigate the mechanisms driving endotheliopathy in diabetes. Our in vitro and in vivo models recapitulate the effects of hyperglycaemia on the vasculature in the clinical setting. RNA-seq data showed differential expression of genes and proteins involved in EC function and angiogenesis were significantly downregulated in DiPS-ECs in comparison to NDiPS-ECs. Moreover, factors involved endothelial-to-mesenchymal transition were increased in DiPS-ECs. DiPS-ECs had significantly reduced impaired tube formation and barrier stability in vitro. DiPS-ECs displayed impaired angiogenic function demonstrated by poor post-transplant engraftment and decreased blood flow recovery (BFR) when injected to the hindlimb of mice following femoral artery ligation. Proteomic and transcriptomic analysis confirmed imbalances in several pathways involved in endothelial function. Pathways involved in metabolism, homeostasis and stress response were found to be underrepresented in DiPS-ECs. RNA-seq revealed differences in the transcriptome of DiPS-ECs. EndMT related gene ontology was enriched in DiPS-ECs. Endothelial specific Roundabout protein 4 (ROBO4) is highly involved in pathways related to angiogenesis, barrier stability and endothelial health. Expression of ROBO4 was found to be impaired in DiPS-ECs and transcriptomic analysis along with in vitro and in vivo studies revealed its importance in vascular specification and angiogenesis. ROBO4 improved ECs function of DiPS-ECs and recovered their lost angiogenic function. Our data suggest that DiPS-ECs carry an imprint of the diabetic milieu which is reflected in their dysfunction. An unregulated EndMT process is proposed to cause a phenotype drift and poor angiogenic functionality observed in DiPS-ECs. ROBO4 seems to act as a fine tuning factor of this process. To the best of our knowledge, we aim to identify and report a novel disease-specific signature in diabetic iPS-ECs. Therefore, our human iPS-ECs model may serve as a valuable tool to study biological pathways and identify new treatments for diabetes-induced endotheliopathy.

Perinatal inflammation is associated with an increased risk of brain injury and neurodevelopmental impairment in preterm infants but the immune mediators driving this association are not well understood. This PhD thesis seeks to further characterise the inflammatory response associated with preterm birth, describe the relationship between perinatal inflammation and white matter development and explore the effect of specific inflammation-associated proteins on the development of human cortical neurons derived from induced pluripotent stem cells (iPSCs). In the first study, I investigated the inflammatory profile at birth in 55 very preterm infants, compared to 59 term-born controls and then used this profile to predict exposure to intrauterine inflammation in the preterm group. Preterm infants had a distinct pro-inflammatory profile in umbilical cord blood at delivery when compared to term-born controls and IL-8 was found to be the strongest predictor of intrauterine inflammation. In the second study, I investigated the association between specific inflammation-associated proteins and white matter microstructure in 71 very preterm infants using structural MRI and diffusion-weighted imaging of the brain. Elevated IL-8 in the first week of life was associated with white matter dysmaturation at term-equivalent age. Following this discovery, I investigated the effect of IL-8 on the maturation and morphology of iPSC-derived cortical neurons and found that exposure was associated with impaired neurite outgrowth. This thesis provides further evidence to support the role of inflammation in the aetiology of preterm brain injury and suggests that IL-8 dysregulation may link systemic inflammation with atypical cortical development and white matter disease in preterm infants.

Survival and persistence of live matter requires certain flexibility to allow multiple adaptations, yet it is grounded on orderly and stringent regulation of its important functions. An astounding example of natural planning is given by mammalian embryological development where a sophisticated specification program enclosed in a single cell is resolved into a diversity of tissues in a mature organism. Tracing back the developmental timeline is a way to deconvolute advanced biological properties that cells acquire through a series of hierarchical differentiation decisions. One of the primary unspecified states spanning a short period between conception and functional commitment of the cell is known as pluripotency. Pluripotency is the ability of a cell to give rise to the derivatives of the three germ layers and germ cells during differentiation. Naturally, pluripotency is restricted to the early embryo only and represented by two morphologically, physiologically and epigenetically sequential stages - naïve (embryonic stem cells, or ESCs) and primed (post-implantation epiblast stem cells, or EpiSCc). Revolutionary technologies of nuclear reprogramming are now able to artificially revert developmental vector and direct differentiated cells towards naïve pluripotency. Reprogrammed cells not only come as a useful object for fundamental research but also as a flexible biological tool for regenerative therapies. Currently reprogramming is moderately efficient, however reliable and safe medical strategies require decent understanding of the physiological and genetic networks underpinning cell identity change. Importantly, the primed pluripotent cell can be reprogrammed too allowing to focus on the first and often subtle cell identity transitions that cells adopt. In my project, I have performed a forward genome-wide CRISPR-gRNA knockout screen to identify genes working as barriers for reprogramming from EpiSCs to ESCs. Having assembled a comprehensive lentiviral library and established a tractable cell system to target the mouse genome, I screened EpiSCs in the reprogramming setup which allowed selection of effective reprogramming antagonists. Here I discover the JAK/STAT3 pathway regulator Socs3 as a key screen hit and describe this gene as a self-sufficient and powerful roadblock between the pluripotent identities. I show how a spectrum of its attenuated activities displayed in naïve pluripotent and reprogramming cells may be functionally linked to reinforced pluripotency. Exploring the interplay between JAK/STAT3 pathway and other major naïve pluripotency genes across naïve, primed and non-pluripotent cell contexts reveals partially conservative mode of action of SOCS3. I demonstrate how SOCS3 may integrate external signalling with endogenous core pluripotency factors and by thereby feed in the naïve circuity supporting it and instructing its establishment. This work helps understand the mechanisms behind reprogramming and biology of pluripotency which potentially can provide insights into consolidated and robust medical cell technologies.

Alzheimer's disease is a progressive neurodegenerative condition that is characterised by distinct neuropathological changes. Within the last decade post mortem human brain samples have been used to show that there are robust epigenetic changes occurring in the brain during disease. However, as these samples are collected shortly after death they are a reflection of only the very end stages of disease. Through the exposure of differentiated adult cells to exogenous reprogramming factors it is now possible to generate induced pluripotent stem cells which have the potential to differentiate into any cell type in the body. Over recent years reseach has moved towards using these stem cells to generate neurons or microglia in order to study diseases of ageing such as Alzheimer's disease. However, there are relatively few epigenetic studies that have been undertaken using induced pluripotent stem cells. As there are global cellular epigenetic changes occurring during the induction of pluripotency and re-differentiation it is critical to ensure we understand the DNA methylation changes occurring during normal neuronal differentiation before using these as a model of Alzheimer's disease or other diseases of ageing. The aim of this thesis is to first characterise the DNA methylation changes that are occurring in neuronal and microglial models that are exposed to AD-relevant exposures such as differentiation and maturation, drug treatment and immune challenge. This will largely be achieved through measuring DNA methylation using the Illumina Infinium HumanMethylationEPIC BeadChip array which provides information on the DNA methylation levels at over 850,000 loci across the genome.

Direct cell reprogramming is a rapidly growing field that has challenged traditional concepts of cellular identity. The expression of Neurogenin 2 (NGN2) results in rapid reprogramming of human pluripotent stem cells (PSCs) into functional excitatory neurons. My lab has previously demonstrated that gene targeting the components of a Tet-On system overexpressing NGN2 into two separate safe harbour sites overcome gene silencing and results in optimised transgene expression in hiPSCs (Opti-Ox), and consequently yields highly homogenous cultures of neurons within less than four days. The mechanisms that mediate this remarkable cellular metamorphosis however remain poorly understood. To explore this, I first sought to establish a protocol for long-term culture of electrophysiologically functional NGN2 induced neurons (iNs). This was achieved by co-culturing with primary rat-derived glial cells, enriched for astrocytes. iNs demonstrated functional activity around two weeks post-induction and by three weeks, formed networks of synchronous bursts that were mediated by glutamatergic AMPA-receptors. Based on these phenotypical hallmarks, I designed a time course-based genomic analysis that investigated the transcriptional and chromatin accessibility states of cells undergoing NGN2 reprogramming. Bulk RNA and ATAC-sequencing were performed at Day 0, 6h, 12h, Day 1, 36h, Day 2, Day 3, Day 4, Day 14 and Day 21 post-NGN2 induction. I also performed a scRNA-seq of the same time points, except for 6- and 36-hours post-induction, to investigate any heterogeneity in the time course and complement findings from the bulk RNA-seq. In order to differentiate direct from indirect NGN2 down-stream effectors, ChIP-seq of NGN2 binding acquired on day 1 after induction was subsequently overlaid with bulk-seq data. In addition, to study the genome-wide effects of astrocyte-enriched rat glia, I performed the same assays on neurons co-cultured with glia at Day 4, 14 and 21. Together, they revealed rapid transcriptional and accessibility changes induced by NGN2 within 6 hours of reprogramming. The subsequent events show a stark similarity to the familiar stages of neurogenesis found in development or conventional differentiation protocols - shutting down of non-neuronal networks, in this case pluripotency, establishment of neuronal commitment in an NSC-like stage by Day 1 post-induction, cell cycle exit by Day 3 or Day 4 and subsequent onset of neuronal differentiation, followed by neuronal maturation. Up until now, this entire process was believed to be a highly homogenous occurrence, but findings from the scRNAseq analysis showed that in addition to glutamatergic neurons, our NGN2 iNeurons are made up of two additional types of neurons: cholinergic neurons with a visceral motor phenotype and neurons with a hybrid profile of cholinergic and glutamatergic transcription. Comparison with neurons co-cultured with glia found an enrichment for synaptic genes and ontologies. Specifically, there was an enrichment for neuronal activity regulated genes in the post-synaptic compartment. Candidate transcription factors crucial to these processes and the states described above were identified from the transcriptional and epigenomic datasets. It is likely that some of these factors could potentially be crucial regulators of neuronal differentiation and function, not only in this protocol, but in development as well. Therefore, one major aim for the future would be to further explore the role these genes play in NGN2 reprogramming through gene knockdown or overexpression experiments along with investigation of their genomic binding sites. Ultimately, this could also lead to the discovery of new reprogramming strategies for producing neurons with enhanced maturity and functionality without the need of human or rodent glia. By uncovering broad, yet essential neuronal processes such as neuronal fate commitment and maturation, the dataset I have generated can be used as a useful resource for studying these processes and designing relevant disease models using this simple, yet robust and efficient protocol for generating functional excitatory human neurons.

The development of targeted therapies has had a significant impact on cancer survival rates. However, targeting cancers that are driven by loss of tumour suppressor genes remains a major challenge. One promising approach to treat these cancers is the exploitation of synthetic lethal interactions. Synthetic lethality describes an interaction between two genes, where loss of one gene alone does not affect viability but loss of both genes induces cell death. Inhibiting the synthetic lethal partner of a tumour suppressor gene should specifically kill tumour cells, and so these represent potential therapeutic targets. However, very few synthetic lethal interactions have been well-established. The aim of this project was to systematically screen for synthetic lethal partners of known tumour suppressor genes. To do so, isogenic human induced pluripotent stem cell lines were generated, each carrying a loss-of-function mutation in a single tumour suppressor gene. These cells have a normal genetic background, thus making it simpler to accurately identify interactions. CRISPR/Cas9 technology was applied as it allows for large-scale, unbiased screening of genetic interactions. A genome-wide guide RNA library was prepared and implemented for knockout screening in the isogenic cell line panel. Analysis was performed to identify genes that were specifically essential for cell fitness/survival in the mutant lines. Particular focus was placed on four tumour suppressor genes that encode subunits of the PBAF/BAF complexes. Approximately 20% of human cancers harbour mutations in subunits of these complexes, so identifying dependencies associated with these could have broad therapeutic potential. Candidate synthetic lethal interactions with these genes were investigated using low-throughput assays in the stem cells and in a cancer cell line. The data obtained suggests that screening in stem cells produces highly variable results. Although potential vulnerabilities associated with all of the tumour suppressor genes were identified, further work is required to validate these and to assess the quality of the results. In addition to genome editing, CRISPR/Cas9 has been adapted as a tool for controlling gene regulation. In collaboration with Dr Louise van der Weyden, I applied this technology to address another challenging area of cancer biology. Metastasis is the main cause of cancer mortality, yet we still have a poor understanding of the genes that control this process. Considering this, an in vivo CRISPR activation screen was performed to identify novel drivers of metastatic colonisation. A mouse melanoma cell line was transduced in vitro with a library designed to up-regulate expression of membrane proteins, which represent ideal drug targets. These cells were then used in an in vivo experimental metastasis assay. Enrichment of guide RNAs in the lungs was assessed to identify genes that increased pulmonary metastatic colonisation when activated. Candidate genes were selected using three analysis strategies, and hits from each were tested. Several genes were successfully validated using the experimental metastasis assay. The most robust hit was studied further to explore its potential as a therapeutic target. Collectively, the work described in this thesis demonstrates how CRISPR/Cas9 screening can be applied in different model systems to study genes that drive cancer and to explore novel therapeutic strategies.

In multiple sclerosis (MS), loss of myelin or demyelination, can be initially restored by oligodendrocyte progenitor cells (OPCs) that migrate to, and proliferate in the site of injury, differentiate into mature oligodendrocytes (OLs) and generate new myelin sheaths, a process termed remyelination. The signals that influence this process in the central nervous system (CNS) of MS patients are still not fully understood, however, recent studies have uncovered an important role for the immune system in promoting remyelination in mice via an enhancement of OPC differentiation. These studies now warrant translational research in humans to investigate the full regenerative potential of immune cells in CNS repair. Based on findings from the mouse that depletion of regulatory T cells (Treg) impairs CNS remyelination and that soluble factors secreted from murine Treg promote OPC differentiation, this PhD Thesis hypothesized that human Treg recapitulate the effect of murine Treg in promoting the differentiation of human OPCs in vitro. To this end, human Treg subsets, including in vitro-induced (iTreg) and naturally-occurring Treg (nTreg) cells were activated in parallel with three non-Treg control populations, total CD4+ T cells that contained a low frequency of nTreg, FOXP3+ T cells that exhibited no suppressive function and whole peripheral blood mononuclear cells (PBMCs) that consisted both of total CD4+ and CD8+ T cells. Conditioned media (CM) from these activated cultures were next harvested and added on human induced pluripotent stem cell (iPSC)-derived mixed neuroglial cells to examine changes in OPC differentiation and proliferation by immunocytochemistry. Soluble factors secreted from activated T cell subsets, all had a trophic influence on OPCs resulting in reduced numbers of O4+ and MBP+ oligodendrocyte lineage cells. This trophic effect was greatly lessened by nTreg, which had no impact on OPC differentiation, but could be clustered into two subgroups of donors, one that induced a high number of mature MBP+ OLs, and one that had no influence on the process. When in culture with glia, activated nTreg cells were permissive to OPC differentiation. When in culture with naive T cells, activated nTreg cells suppressed the production of factors that inhibited OPC differentiation as shown by the treatment of glia with suppression assay-CM. Multivariate analyses of T cell secretion data confirmed the relatedness of iTreg and nTreg, and differentiated these cells from other non-Treg populations. In addition, iTreg cells were found to be enriched for proteins that hinder OPC differentiation, whereas nTreg cells exhibited low secretion of such proteins, and variable secretion of proteins with the capacity to promote this process. These findings establish the phenotypic and functional integrity of regulatory features as both being critical determinants of OPC differentiation. The in vitro studies on the interaction of human T cell subsets with oligodendrocytes presented in this Thesis, advance our knowledge about the influence of human Treg on OPC proliferation and differentiation. These are critical processes required for the efficient regeneration of the CNS myelin sheath, which occurs at significantly slower rates in patients with MS. The results of this Thesis demonstrate that human Treg are permissive to OPC differentiation and protective to this process via the suppression of effector T cell responses. This new knowledge could prove valuable for therapies that aim to enhance Treg function in MS.

Mutations in the gene encoding the bone morphogenetic protein type 2 receptor (BMPR2) are the most common genetic cause of heritable pulmonary arterial hypertension (PAH). However, given the reduced penetrance of BMPR2 mutations in affected families, a major outstanding question is the identity of additional factors or pathways that are responsible for the manifestation of clinical disease. Furthermore, limited human tissue is available for study and usually only from patients with end-stage disease, making it difficult to understand how PAH is established and progresses. Alternative human models of PAH are therefore required. This thesis describes the characterisation of the first human iPSC-derived smooth muscle cell (iPSC-SMC) model of PAH and elucidates the role of BMPR2 deficiency in establishing PAH-associated phenotypes in iPSC-derived SMCs. To achieve this, I used CRISPR-Cas9 gene editing to generate wild-type and BMPR2+/- iPSC lines with isogenic backgrounds which were subsequently differentiated into lineage-specific iPSC-SMCs that displayed a gene expression profile and responses to BMP signalling akin to those present in distal pulmonary artery smooth muscle cells (PASMCs). Using these cells, I found that the introduction of a single BMPR2 mutation in iPSC-SMCs was sufficient to recapitulate the pro-proliferative and anti-apoptotic phenotype of patient-derived BMPR2+/- PASMCs. However, acquisition of the mitochondrial hyperpolarisation phenotype was enhanced by inflammatory signalling and required an interaction between BMPR2 mutations and environmental stimuli provided by exposure to serum over time. Furthermore, I showed that BMPR2+/- iPSC-SMCs had an altered differentiation state and were less contractile compared to wild-type iPSC-SMCs, phenotypes which have not been observed previously in PAH-derived PASMCs. Finally, RNA sequencing analysis identified genes that were differentially expressed between wild-type and BMPR2+/- iPSC-SMCs and may hence provide further insights into PAH pathobiology. The iPSC-SMC model described in this study will be useful for identifying additional factors involved in disease penetrance and for validating therapeutic approaches that target BMPR2.

Genetic associations have been discovered for many human complex traits, and yet for most associated loci the causal variants and molecular mechanisms remain unknown. Studies mapping quantitative trait loci (QTLs) for molecular phenotypes, such as gene expression, RNA splicing, and chromatin accessibility, provide rich data that can link variant effects in specific cell types with complex traits. These genetic effects can also now be modeled in vitro by differentiating human induced pluripotent stem cells (iPSCs) into specific cell types, including inaccessible cell types such as those of the brain. In this thesis, I explore a range of approaches for using QTLs to identify causal variants and to link these with molecular functions and complex traits. In Chapter 2, I describe QTL mapping in 123 sensory neuronal cell lines differentiated from human iPSCs. I observed that gene expression was highly variable across iPSC-derived neuronal cultures in specific gene categories, and that a portion of this variability was explained by commonly used iPSC culture conditions, which influenced differentiation efficiency. A number of QTLs overlapped with common disease associations; however, using simulations I showed that identifying causal regulatory variants with a recall-by- genotype approach in iPSC-derived neurons is likely to require large sample sizes, even for variants with moderately large effect sizes. In Chapter 3, I developed a computational model that uses publicly available gene expression QTL data, along with molecular annotations, to generate cell type-specific probability of regulatory function (PRF) scores for each variant. I found that predictive power was improved when the model was modified to use the quantitative value of annotations. PRF scores outperformed other genome-wide scores, including CADD and GWAVA, in identifying likely causal eQTL variants. In Chapter 4, I used PRF scores to identify relevant cell types and to fine map potential causal variants using summary association statistics in six complex traits. By examining individual loci in detail, I showed how the enrichments contributing to a high PRF score are transparent, which can help to distinguish plausible causal variant predictions from model misspecification.

Parkinson’s disease (PD) is the second most common age-related neurodegenerative condition. It is neuropathologically characterised by the presence of Lewy pathology and the degeneration of the midbrain dopaminergic neurons from the substantia nigra pars compacta. Lewy pathology mainly consists of filamentous aggregated alpha-synuclein and familial forms of PD can be caused by genetic alternations in the SNCA gene encoding alpha-synuclein. Alpha-synuclein is primarily localised to neuronal presynaptic terminals and has been implicated in the maintenance of synaptic function. Studies have proposed that it regulates the docking, fusion, clustering and trafficking of neurotransmitter-loaded presynaptic vesicles. Nowadays, it is possible to model PD in vitro by obtaining adult somatic cells from patients, reprogramming them into induced pluripotent stem cells (iPSCs), and differentiating iPSCs into neurons. For this project, iPSCs derived from two PD patients, one harbouring the A53T SNCA mutation, the other a SNCA triplication, and three healthy individuals, were employed. In the initial stage, I optimised a neuronal differentiation protocol originally developed for human embryonic stem cells to produce neurons belonging to two distinct brain regions affected in PD, the forebrain and midbrain, from the available human iPSC lines. Next, I assessed the maturation of the generated neurons over time using protein expression and electrophysiological techniques. Finally, I examined PD-related phenotypes such as alpha-synuclein aggregation and release, susceptibility to cell death, and the redistribution of presynaptic proteins. All the iPSC lines used gave rise to forebrain and midbrain neuronal cultures. Maturation was similar across lines, as no consistent differences were observed in the changes of the expression of 4 repeat tau isoforms, presynaptic protein levels or electrophysiological properties over time. However, the emergence of astrocytes varied between cultures derived from distinct iPSC lines. No robust differences in alpha-synuclein release and susceptibility to cell death were detected between patient- and control-derived neurons. Apart from the presence of larger alpha-synuclein-positive puncta in patient-derived neurons, no other signs of alpha-synuclein aggregation were detected. Despite this, midbrain patient-derived neurons with a SNCA triplication exhibited a significant redistribution of presynaptic protein VAMP-2/synaptobrevin-2, which interacts with alpha-synuclein, relative to controls.

Chemical modification of the cytosine base via the addition of a methyl group to form 5-­‐methylcytosine (5-­‐mC) is a well-­‐studied example of an epigenetic mark, which contributes to regulation of gene expression, chromatin organisation and other such cellular processes without affecting the underlying DNA sequence. In recent years it was shown that 5-­‐mC is not the only DNA modification found within the vertebrate genome. 5-­‐hydroxymethylcytosine (5-­‐hmC) was first described in 1952 although it wasn’t until 2009 when it was rediscovered in mammalian tissues that it sparked intense interest in the field. Research has found that unlike the 5-­‐mC base from which it is derived, 5-­‐hmC displays variable levels and patterns across a multitude of tissue and cell types. As such the patterns of these DNA modifications can act as an identifier of cell state. This thesis aims to characterize the methyl and hydroxymethyl profiles of induced pluripotent stem cells (iPSCs), derived from control mouse embryonic fibroblast cell line (p53-­‐/-­‐) as well as and methylation hypomorphic (p53-­‐/-­‐, Dnmt1 -­‐/-­‐) mutant cell lines. As such both somatic cells were subject to reprogramming with Yamanaka factors (Oct4, cMyc, Klf4 and Sox2) via the piggyback transposition technique. Successful reprogramming was confirmed by a number of techniques and outcomes, including the de novo expression of a number of key pluripotency related factors (Nanog, Sall4 and Gdf3). Reprogrammed cells were then analysed for transcriptomic changes as well as alterations to their methyl and hydroxymethyl landscapes that accompany reprogramming. Through this work I have shown that the reprogramming of MEF derived cell lines results in a global increase in 5-­‐hmC for both p53-­‐/-­‐ and (p53-­‐/-­‐, Dnmt1 -­‐/-­‐) hypomorphic mutant cell lines – possibly through the reactivation of an alternative form of DNMT1. I demonstrate by both antibody based dot blot assay and genome wide sequencing that the reprogramming of the (p53-­‐/-­‐, Dnmt1 -­‐/-­‐) somatic cells towards a pluripotent state brings about an increase in methylation levels within the cells. This latter observation may indicate that the reprogramming of the cells is driving them towards a more wild type phenotypic state. My studies suggest that lack of DNMT1 function is not a barrier to reprogramming of somatic cells.

Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disorder characterised by selective loss of upper and lower motor neurons (MNs). Recently, the GGGGCC (G4C2) hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72) has been identified as the most common genetic cause of ALS, highlighting the importance of studying the pathogenic mechanisms underlying this mutation. Accumulating evidence implicates that ALS is a multisystem and multifactor disease. Specifically, non-neuronal cells, astrocytes in particular, are also affected by toxicity mediated by ALS-related mutations, and they can contribute to neurodegeneration, suggesting astrocytes as a key player in ALS pathogenesis. Here, a human induced pluripotent stem cells (iPSCs)-based in vitro model of ALS was established to investigate the impact of the C9orf72 mutation on astrocyte behaviour—both cell- and non-cell autonomous. Work in this study shows that patient iPSC-derived astrocytes recapitulate key pathological features associated with C9orf72-mediated ALS, such as formation of G4C2 repeat RNA foci, production of dipeptide repeat (DPR) proteins and reduced viability under basal conditions compared to controls. Moreover, C9orf72 mutant astrocytes in co-culture result in reduced viability and structural defects of human MNs. Importantly, correction of the G4C2 repeat expansion in mutant astrocytes through targeted gene editing reverses these phenotypes, strongly confirming that the C9orf72 mutation is responsible for the observed findings. Altogether, this iPSC-based in vitro model provides a valuable platform to gain better understandings of ALS pathophysiology and can be used for future exploration of potential therapeutic drugs.

Parkinson's disease (PD) primarily manifests as loss of motor control through the degeneration of nigrostriatal dopaminergic neurons. The microtubule-associated protein tau (MAPT) locus is highly genetically associated with PD, wherein the H1 haplotype confers disease risk and the H2 haplotype is protective. As this haplotype variation does not alter the amino acid sequence, disease risk may be conferred by altered gene expression, either of total MAPT or of specific isoforms, of which there are six in adult human brain. To investigate haplotype-specific control of MAPT expression in the neurons that die in PD, induced pluripotent stem cells (iPSCs) from H1/H2 heterozygous individuals were differentiated into dopaminergic neuronal cultures that expressed all six mature isoforms of MAPT after six months' maturation. A reporter construct using the human tyrosine hydroxylase locus was also generated to identify human dopaminergic neurons in mixed cultures. Haplotype-specific differences in the inclusion of exon 3 and total MAPT were observed in iPSC-derived dopaminergic neuronal cultures and a novel variant in MAPT intron 10 increased the inclusion of exon 10 by two-fold. RNA interference tools were generated to knockdown total MAPT or specific isoforms, wherein knockdown of the 4-repeat isoform of tau protein increased the velocity of axonal transport in iPSC-derived neurons. MAPT knockdown also reduced p62 levels, suggesting an impact of tau on macroautophagy, likely through altered axonal transport. These results demonstrate how variation at a disease susceptibility locus can alter gene expression, thereby impacting on neuronal function.

Induced pluripotent stem cells (iPSCs) are generated by reprogramming somatic cells to an embryonic state. Therefore iPSCs represent an extremely valuable tool for modelling disease and organ toxicity, with enormous potential in veterinary medicine. Several equine diseases are currently untreatable and can result in euthanasia on medical grounds. In contrast to humans, in vitro models for cellular research in equine do not exist. Therefore it has been necessary to explore the use of stem cells in constructing cell based equine models. Pluripotent stem cell populations are of great interest in this field given their ability to form the three germ layers found in the developing embryo. While a promising notion, the isolation of equine embryonic stem cells has thus far proved elusive and therefore it has been necessary to explore other pluripotent stem cell populations. A very limited number of induced PSC lines have so far been generated from equine fibroblasts but studies in humans showed that other cell types such as keratinocytes were more amenable to reprogramming and generated iPSCs with much higher efficiency; whether this may be also the case in other species has not been investigated. Moreover, iPSC lines reported so far from domestic species, including the horse, depended on complex culture conditions for growth, including feeder layers and media supplementation with several growth factors. Although a promising alternative to fibroblast for generation of induced pluripotent stem cells there is dearth in literature on equine keratinocyte culture techniques. In this work I am reporting a novel approach to generate equine iPSCs lines from keratinocytes. Skin biopsies were used to derive keratinocyte cultures. The three dimensional culture systems were developed for robust culture of equine keratinocytes. These cells were then transduced with retroviral constructs coding for murine Oct-4, Sox-2, c-Myc and Klf-4 sequences, following the original Yamanaka protocol. Following transduction, tight cell colonies with sharp boundaries staining positive for alkaline phosphatase resembling previously reported human iPSCs were generated. The reprogrammed cells were successfully maintained in feeder free and serum free conditions with LIF supplementation. Immunochemistry and qPCR analyses revealed the equine iPSCs lines expressed pluripotency markers expressed in equine embryonic stages including, OCT4, SOX2, SSEA1, LIN 28, NANOG, REX1 and DNMT3B. Equine iPSCs were able to form embryoid bodies and differentiate into derivatives of the three germ layers in vitro. Equine iPSCs were pluripotent in vivo as demonstrated by the formation of teratoma consisting of tissue derivatives of all three lineages such as bone, cartilage, pulmonary epithelium and mature neurons in SCID mice. Importantly, equine iPSCs should not only have the ability to differentiate in a non-directed manner. Therefore, the ability for efficient and directed cellular differentiation was analysed. Equine iPSCs were successfully induced to differentiate into neurospheres forming extensive neuronal projections and synapses. Equine iPSCs were differentiated to neurons using a novel and robust approach. The neurons expressed FOXG1, TUBB3 at induction before ISL1 up regulation, a potent and specific inducer of motor neurons, during terminal differentiation. The neurons tested could fire multiple action potentials and also induce TTX –sensitive action potentials. The iPSC line that showed in vivo differentiation in bone and cartilage was tested for directed differentiation into bone and results were compared to equine mesenchymal stem cells. This study provides the first demonstration of the potential of iPSCs in equine biomedicine. The ability to derive iPSC cells capable of direct differentiation in vitro opens the way for new and exciting applications in equine regenerative medicine.

Amyotrophic Lateral Sclerosis (ALS) is an incurable late onset neurodegenerative disorder characterised by the specific loss of motor neurones (MNs). It has been recently demonstrated that Transactive response DNA-binding protein (TDP-43) is the dominant disease protein in both ALS and a sub-group of frontotemporal lobar degeneration (FTLDTDP). Moreover, the identification of TARDBP mutations in familial ALS confirms a mechanistic link between the observed mis-accumulation of TDP-43 and neurodegeneration but also provides an opportunity to establish an in vitro platform to model these diseases, based on patient-derived induced pluripotent stem cells (iPSCs). This study presents the optimization of an iPSC-based platform to study the consequences of TDP-43 M337V mutation in human functional populations of MNs and astrocytes in isolation as well as in co-culture. To develop this platform, two protocols to differentiate patient-derived iPSCs into functional MNs and astrocytes were first optimized, and the obtained cellular populations were then used to characterize the behaviour of mutant TDP-43 and its effect on the different cell types. This study show that it is possible to use iPSC-based platforms to recapitulate in vitro key aspects of TDP-43 proteinopathies such as MN cell autonomous toxicity and TDP-43 accumulation, but they can also be used to highlight previously unrecognised disease specific mechanisms and to test novel therapeutic approaches. Moreover, by performing co-culture experiments it was possible to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of co-cultured neurons. This iPSC-based platform represents an in vitro model to study both the effect of somatic mutations on isolated patient-specific cultures, but also to investigate cellular autonomy and neurodegeneration in the context of TDP-43 proteinopathies.

Embryonic stem cells (ESCs) are pluripotent cells derived from the early embryo and are able to differentiate into cells belonging to the three germ layers. They are a valuable tool in research and for clinical use, but their applications are limited by ethical and technical issues. In 2006 a breakthrough report described the generation of induced pluripotent stem cells (iPSCs). IPSCs are ESC-like cells generated from somatic cells by forcing the ectopic expression of specific transcription factors. This circumvents the ethical issues about the use of embryos in research and provides multiple opportunities to understand the mechanisms behind pluripotency. The aim of this project was to generate sheep iPSCs and characterise them. In order to learn the technique I initially repeated the original iPSC methodology: the putative mouse iPSCs I have generated display a morphology typical of ESCs, characterised by a high nuclear to cytoplasmic ratio, and form colonies with neat edges and smooth domes. These cells are positive to Nanog, a marker of pluripotency, and can give rise to cells belonging to the mesodermal and the ectodermal lineages when differentiated in vitro. Since the main aim of the thesis was the derivation of sheep pluripotent cells, once established the protocol in mouse, I then moved to the generation of ovine iPSC colonies. The cells I have generated have a morphology similar to that of mouse ESCs, express markers of pluripotency such as alkaline phosphatase and Nanog and can differentiate in vitro and in vivo into cells belonging to the three germ layers. Additionally, these ovine iPSCs can contribute to live born chimeric lambs, although at low level.

Mouse models have been classically used to study human neurodevelopmental disorders. More recently, human stem cell models have advanced our ability to model unique aspects of human brain. While mice are able to recapitulate many aspects of human brain development, human cortex is vastly expanded compared to mice. This dissertation will show how mouse models and human iPSC models can be used in combination to generate increased understanding of the pathology and molecular mechanism of several human brain disorders.First, we studied Joubert syndrome. KIAA0753 is one of at least 35 Joubert causal genes. We describe four new patients with five novel KIAA0753 variants and study these variants in vitro. The variants produce truncated KIAA0753 protein products that are not competent to promote primary ciliogenesis. Patient fibroblasts and Kiaa0753 knockout 3T3 cells have a loss of primary cilia and subsequent deficit in SHH and WNT pathway signaling. Utilizing these complementary cell models allowed us to demonstrate the pathogenicity of specific human variants.Second, we identified a developmental brain disorder caused by human variants in CAMSAP1. Patients present with a characteristic craniofacial appearance and numerous structural brain abnormalities including microcephaly. We generated a Camsap1 null mouse allele which exhibits increased perinatal mortality but showed no severe structural brain abnormalities. We used human induced pluripotent stem cells (iPSCs) obtained from a CAMSAP1 patient to make neural rosette structures which display abnormal morphology, decreased proliferation, and premature neuronal differentiation. These cellular phenotypes suggest a neural progenitor defect. Mouse models showed us that Camsap1 is expressed in brain and required for postnatal survival, and adding a human iPSC model allowed us to demonstrate the effects of CAMSAP1 variants on early forebrain cell development.Finally, we studied the impact of sphingolipid biosynthesis on human brain development. SMPD4 variants cause a severe developmental brain disorder including microcephaly and cerebellar hypoplasia. We hypothesized that the role of SMPD4 in producing ceramide is important for making primary cilia. We found that Smpd4 null mice have cerebellar hypoplasia due to failure of Purkinje cell development. By using human iPSC models we were able to show that the microcephaly is likely due to a loss of neural progenitor cells, which exhibit cell death and decreased proliferation in vitro. We explored the role of primary cilia in these models and found that although Smpd4 null mice appear unaffected, human iPSC models have shortened and bulbous primary cilia which is rescued by adding exogenous ceramide. We found evidence for disrupted WNT pathway signaling. We suggest that during brain development, SMPD4 provides ceramide in an organelle-specific manner, regulating the cell cycle and allowing primary cilia to grow properly.In summary, common signatures such as primary cilia morphology and neural progenitor proliferation are impacted by patient variants in brain development genes. The power of combining mouse and human models lies in its potential to uncover the molecular roles of these genes. We have utilized this combination to advance our knowledge of the roles of KIAA0753, CAMSAP1, and SMPD4 genes for human brain development.

Down syndrome (DS) is caused by triplication of human chromosome 21 (HSA21) and is the most common genetic form of intellectual disability. It is unknown precisely how triplication of HSA21 results in the intellectual disability, but it is thought that the global transcriptional dysregulation caused by trisomy 21 perturbs multiple aspects of neurodevelopment that cumulatively contribute to its etiology. While the characteristics associated with DS can arise from any of the genes triplicated on HSA21, in this work we focus on oligodendrocyte transcription factor 2 (OLIG2). The progeny of neural progenitor cells (NPCs) expressing OLIG2 are likely to be involved in many of the cellular changes underlying the intellectual disability in DS. To explore the fate of OLIG2+ neural progenitors, we took advantage of two distinct models of DS, the Ts65Dn mouse model and induced pluripotent stem cells (iPSCs) derived from individuals with DS. Our results from these two systems identified multiple perturbations in development in the cellular progeny of OLIG2+ NPCs. In Ts65Dn, we identified alterations in neurons and glia derived from the OLIG2 expressing progenitor domain in the ventral spinal cord. There were significant differences in the number of motor neurons and interneurons present in the trisomic lumbar spinal cord depending on age of the animal pointing both to a neurodevelopment and a neurodegeneration phenotype in the Ts65Dn mice. Of particular note, we identified changes in oligodendrocyte (OL) maturation in the trisomic mice that are dependent on spatial location and developmental origin. In the dorsal corticospinal tract, there were significantly fewer mature OLs in the trisomic mice, and in the lateral funiculus we observed the opposite phenotype with more mature OLs being present in the trisomic animals. We then transitioned our studies into iPSCs where we were able to pattern OLIG2+ NPCs to either a spinal cord-like or a brain-like identity and study the OL lineage that differentiated from each progenitor pool. Similar to the region-specific dysregulation found in the Ts65Dn spinal cord, we identified perturbations in trisomic OLs that were dependent on whether the NPCs had been patterned to a brain-like or spinal cord-like fate. In the spinal cord-like NPCs, there was no difference in the proportion of cells expressing either OLIG2 or NKX2.2, the two transcription factors whose co-expression is essential for OL differentiation. Conversely, in the brain-like NPCs, there was a significant increase in OLIG2+ cells in the trisomic culture and a decrease in NKX2.2 mRNA expression. We identified a sonic hedgehog (SHH) signaling based mechanism underlying these changes in OLIG2 and NKX2.2 expression in the brain-like NPCs and normalized the proportion of trisomic cells expressing the transcription factors to euploid levels by modulating the activity of the SHH pathway. Finally, we continued the differentiation of the brain-like and spinal cord-like NPCs to committed OL precursor cells (OPCs) and allowed them to mature. We identified an increase in OPC production in the spinal cord-like trisomic culture which was not present in the brain-like OPCs. Conversely, we identified a maturation deficit in the brain-like trisomic OLs that was not present in the spinal cord-like OPCs. These results underscore the importance of regional patterning in characterizing changes in cell differentiation and fate in DS. Together, the findings presented in this work contribute to the understanding of the cellular and molecular etiology of the intellectual disability in DS and in particular the contribution of cells differentiated from OLIG2+ progenitors.

Embryogenesis is a complex process orchestrated through local morphogen gradients and physical constraints that give rise to the three germ layers. In vitro models of embryogenesis have been demonstrated by treating pluripotent stem cells in adherent or suspension culture with soluble morphogens and small molecules, which leads to tri-lineage differentiation. However, treatment with exogenous agents override the subtle spatiotemporal changes observed in vivo that ultimately underly the human body plan. In this thesis, we demonstrate how deformable hydrogels for pluripotent stem cell culture catalyse gastrulation-like events using materials alone. Micro-confinement through soft lithography enhances cell-cell adhesion and proliferation within the colony, where stress at the interface leads to mechanosensing mediated nuclear shape changes, epithelial to mesenchymal transition, and emergence of defined patterns of primitive streak containing SOX17+ T/BRACHYURY+ cells. Immunofluorescence staining, transcript analysis, and the use of pharmacological modulators reveal a role for mechanotransduction-coupled non-canonical wingless-type (Wnt) signalling and YAP1 signalling dynamics in promoting epithelial to mesenchymal transition at the interface, and multilayered organization within the colonies. These microscale gastruloids were removed from the substrate and grown in suspension culture. Rather than uniform growth as observed using traditional embryoid body culture, the gastruloids undergo multi-lobed outgrowth with evidence of further differentiation both in vitro and in vivo. Encapsulating the gastruloids into several hydrogel biomaterials indicates that materials properties will influence the interfacial positioning of primitive streak, which may prove a useful strategy to further direct differentiation. Together, this thesis demonstrates how materials alone can nurture embryonic gastrulation, with defined microenvironmental cues that mimic pre-streak events, thereby providing an in vitro model of early development. This work provides a new tool to link the biophysical and biochemical parameters of the local microenvironment to cell and tissue morphogenesis for fundamental studies. Using materials to guide morphogenesis in the laboratory provides an approach for directing differentiation, towards new materials-directed organoid models for tissue engineering and regenerative medicine.

iPSCs are conventionally generated under constitutive promoters for a constant expression of pluripotency factors, yet the yield of reprogrammed cells is low. Since somatic cells are entrained in a circadian rhythm that regulates their metabolic and physiological functions, we hypothesize that the expression of the reprogramming factors should be done under circadian rhythm instead of a continuous expression. Doing this might hasten the process and improve the yield of iPSCs. To test this, I prepared plasmid constructs with the OSKM reprogramming cassette attached downstream of the circadian promoters Per2 or Bmal1. However, I could not obtain successful midipreps of these plasmids due to time constraints. Nevertheless, iPSCs were generated from fibroblasts with plasmids containing the OSKM genes under the constitutive LTR promoter. This independent experiment helped me become familiar with the process of virus generation and transduction and to determine the optimum seeding density to achieve iPSC formation from fibroblasts.

White matter disorders constitute several severe neurological diseases including multiple sclerosis and leukodystrophies. In this latter category many of these pediatric genetic myelin disorders have established genetic etiologies and can be effectively modeled a number of spontaneous mouse mutants and transgenics. In spite of these investigational assets few disease-modifying therapies exist to replace lost or dysfunctional myelin.The jimpy mouse is the best characterized leukodystrophy model. It contains a single point mutation the proteolipid protein 1 (PLP1) and genuinely recapitulates many hallmarks of Pelizaeus-Merzbacher Disease (PMD), including loss of myelinating oligodendrocytes, severe neurological deficits, and early mortality. Here we use this mouse model to establish scalable populations of induced pluripotent stem cell (iPSC)-derived mutant oligodendrocyte precursor cells (OPCs) to precisely parse the cellular and molecular phenotypes in the oligodendrocyte lineage over a defined developmental window in vitro. We then overcome these phenotypes using high-throughput chemical screening, identifying Ro 25-6981 as a potent modulator of jimpy oligodendrocyte loss through a non-annotated, unfolded protein response (UPR)-mediating mechanism. While Ro 25-6981 showed broad effects to restore mutant oligodendrocytes in multiple, orthogonal contexts including in PMD patient 3D spheroid cultures and in jimpy mice, in vivo, it was unable to restore stable myelination, revealing a previously unappreciated second pathological phase in PMD. Furthermore this work informs future studies on the oligodendrocyte lineage, defining specific nodes of therapeutic intervention during oligodendrogenesis and myelinationIn parallel we also demonstrate that directly targeting the disease-causative gene in PMD may represent a highly effective, clinically translatable therapeutic strategy for patients. Specifically we used CRISPR to knockout Plp1 in jimpy mice and demonstrate a rescue of all relevant disease phenotypes through rigorous lifespan, biochemical, and functional assessment. Additionally we show multi-month lifespan restoration using clinically-relevant antisense oligonucleotides (ASOs) delivered to postnatal jimpy mice. Together this data establishes strong pre-clinical data and justifies a clinical trial in human PMD patients.Collectively these studies establish a robust drug-discovery pipeline for genetic myelin disorders and validate a new therapeutic strategy for the treatment of PMD.

Human induced pluripotent stem cells (hiPSCs) provide a unique opportunity to study the human specific aspects of human disorders such as Alzheimer’s disease (AD). However, in vitro systems are inherently artificial and lack the complexity of the in vivo environment. Here we demonstrate how chimeric disease modeling, where we transplanted mixed hiPSC-derived neurons (MNs), comprised primarily of excitatory neurons, and enriched inhibitory neurons (INs) into the mouse hippocampus, allow us to model apoE4 toxicity in different subtypes of human neurons within the in vivo environment. Seven to eight months after transplantation, hiPSC-derived neurons survive and functionally integrate into the mouse brain. However, unlike apoE3 knock-in (apoE3-KI) and apoE4-KI mouse neurons, hiPSC-derived neurons generate A aggregates, with INs generating significantly less than MNs. ApoE4/4 mouse microglia display significant deficits in their ability to phagocytose A aggregates generated by hiPSC-derived neurons, resulting in an increase in the number of aggregates in the apoE4/4 mouse brain. Interestingly, apoE4/4 hiPSC-derived INs are selectively vulnerable to the toxicity of the apoE4/4 brain and display elevated tau-phosphorylation relative to MN transplants. This novel in vivo chimeric model for apoE4/4 toxicity displays human-specific pathological features and allows us to identify how the cellular source of apoE4 relates to pathological response in different neuronal subtypes in the course of Alzheimer’s disease.

Cyclin-dependent kinase-like 5 (CDKL5) syndrome is a neurodevelopmental disorder characterized by early-onset intractable seizures, mental retardation, and hand stereotypies. Studies have shown that loss of CDKL5 protein function is responsible for aberrations in neuronal morphogenesis, and decreased synaptogenesis. In order to understand the effects of CDKL5 mutations on human neural disease pathology, we utilized induced pluripotent stem cell (iPSC) technology to generate human iPSC-derived neurons from CDKL5 patients. Furthermore, to circumvent in vitro iPSC disease modeling limitations and to take advantage of an in vivo brain environment, we integrated these two systems by engrafting human iPSC-derived neural progenitor cells (NPCs) into immunodeficient mice. Here, we report defects in neuronal morphology in CDKL5- mutant neurons differentiated in vitro that are retained in human neurons differentiated in a mouse brain. Furthermore, we found that using a histone deacetylase inhibitor, LMK235, specific to histone deacetylase 4 (HDAC4), was able to rescue defects in synaptogenesis and neural electrical activity in CDKL5 iPSC-derived neurons in vitro. Ultimately, we hope our work will provide further insights into CDKL5 disease pathology and accelerate the development of treatments for this complex neurodevelopmental disorder.

The preoptic-anterior hypothalamus (PO/AH) is a major neural area for sensing body temperature and integrating this information with afferent inputs from peripheral thermoreceptors. The PO/AH contains both warm sensitive neurons and various types of temperature insensitive neurons. These neurons form synaptic networks that control different physiological and behavioral thermoregulatory responses. The present series of experiments focused on the cellular mechanisms that account for neuronal thermosensitivity and the types of synapses that constitute PO/AH neuronal networks. Intracellular recordings were made from neurons in rat hypothalamic tissue slices. Current clamp recordings measured the resting membrane potentials and postsynaptic potentials during either temperature changes or manipulations of synaptic activity. Similarly, voltage clamp recordings measured resting and postsynaptic currents during these same conditions. Mechanisms of neuronal thermosensitivity. PO/AH warm sensitive neurons increase their firing rates with increasing temperatures, while the firing rates of temperature insensitive neurons remain relative constant. Previous studies suggested that the main mechanism for neuronal warm-sensitivity is a thermally-induced depolarization due to inward cationic (Na+ and Ca++) currents. One often-cited study (Kiyohara at al., 1990), in particular, indicated that warm sensitive neurons possess a tetrodotoxin-sensitive resting Na+ current that accounts for their thermosensitivity. The present study examined the effects of temperature on the resting membrane potentials and inward currents in different types of PO/AH neurons. Similar responses were observed in both warm sensitive and temperature insensitive neurons. All neurons displayed slight depolarization during temperature increases and hyperpolarization during temperature decreases; and there was no correlation between neuronal (firing rate) thermosensitivity and resting membrane potential thermosensitivity. Also, all neurons displayed similar thermally-induced changes in their resting currents. These currents increased slightly during tissue warming and decreased during tissue cooling; and again, there was no correlation between neuronal thermosensitivity and resting current thermosensitivity. In addition, the Na+ channel blocker, tetrodotoxin (TTX), reduced current thermosensitivities in both temperature insensitive neurons and warm sensitive neurons. These results suggested that thermosensitive resting membrane potentials, resting currents, as well as persistent, TTX-sensitive sodium currents are not the mechanisms for neuronal warm-sensitivity. Synaptic characteristics. The second series of experiments examined the types of synapses that occur within the PO/AH neuronal network. Voltage clamp experiments measured the excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) for the different neuronal types. EPSCs and IPSCs exist in both warm sensitive and temperature insensitive neurons; however, warm sensitive neurons have more IPSCs and less EPSCs than the different groups of temperature insensitive neurons. These experiments also examined the neurotransmitters responsible PO/AH synaptic activity. Glutamate caused most of the EPSCs, and GABA (i.e, GABAA receptor) caused most of the IPSCs. Kynurenic acid (a broad spectrum glutamate receptor antagonist) blocked most EPSCs, and bicuculline methiodide (GABAA receptor antagonist) blocked almost all IPSCs. Moreover, TTX attenuated most synaptic currents in warm sensitive neurons and temperature insensitive neurons; and those synaptic currents that were insensitive to TTX were completed blocked by Na+-free medium. This suggests that the recorded postsynaptic activity was due to neurotransmitter released in response to membrane depolarization in presynaptic fibers.